Manipulation of plant polysaccharide synthases

ABSTRACT

The present invention provides compositions and methods for manipulation of plant polysaccharides and plant polysaccharide synthases. Compositions include novel nucleotide sequences encoding polysaccharide synthases polypeptides, and biologically active variants thereof. Further provided are methods for polysaccharide manipulation using the sequences disclosed herein. One method comprises stably incorporating into the genome of a plant cell, a nucleotide sequence of the present invention operably linked to a heterologous promoter and regenerating a stably transformed plant that expresses the nucleotide sequence. Methods for enhancing digestibility of plants, improving gum extractability from plants, and altering plant growth are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Application SerialNo. 60/325,614 filed Sep. 27, 2001, which is herein incorporated byreference.

FIELD OF THE INVENTION

[0002] The present invention relates to polysaccharide production inplants through alteration of the polysaccharide synthesis pathways.

BACKGROUND OF THE INVENTION

[0003] Cereals constitute a major portion of human nutrition because ofthe polysaccharides the plants produce. Annually, over one billion tonsof cereal grains are harvested, and half the calories consumed by humansare from rice and wheat alone. In addition, grazing animals consume vastamounts of grasses. Although cellulose is the primary polysaccharide ofplants, plant cell walls also contain hemicelluloses and pectins(Carpita (1996) Annu. Rev. Plant Physiol. Plant Mol. Biol. 47:445-476).

[0004] Plant growth is determined by concerted synthesis of cell wallpolymers, such as hemicelluloses and pectins. Thus, increased synthesisof one cell wall polymer is expected to cause an increase in thesynthesis of the other polymers as well. Increased production of a plantpolysaccharide generally accelerates plant growth. Conversely, decreasedproduction of a plant polysaccharide generally inhibits the synthesis ofother cell wall polymers and slows plant growth.

[0005] Mature plant cells generally contain about 30-40% hemicellulose.In monocot species, arabinoxylan (also referred to asglucurono-arabinoxylans or pentosan) is the main component ofhemicellulose in the cell wall. In contrast, dicot cell walls containxyloglucan as the primary hemicellulosic polymer (Carpita (1996) Annu.Rev. Plant Physiol. Plant Mol. Biol. 47:445-476).

[0006] Arabinoxylans are anti-nutritional components of animal feed, yetthese polymers constitute 45-65% of the plant cell wall. Arabinoxylansabsorb large amounts of water thus increasing the viscosity of the chymeand sequestering other digestible nutrients away from the digestiveenzymes (WO 99/67404). In addition, the increased viscosity of the chymeresults in sticky feces that contribute to animal hygiene and entericdisturbance problems for the livestock producer (Selinger et al. (1996)Anaerobe 2:263-284). Therefore, in certain circumstances, it would bedesirable to lower the concentration of arabinoxylans in plants.

[0007] However, dietary fiber, particularly arabinoxylan, reducescholesterol and low density lipoprotein levels in humans (WO 99/67,404).In breadmaking, bread quality depends heavily on the consistency of thedough. Dough that lacks viscosity alters the crumb structure of thebread and decreases the volume of bread produced. Arabinoxylan providesthe viscous properties of dough (Girhammar et al. (1995) FoodHydrocolloids 9:133-140). Additionally, industries use isolatedarabinoxylan preparations as thickeners, emulsifiers, or stabilizers infood, cosmetics, and pharmaceuticals. Therefore, in certaincircumstances, it would be desirable to increase the concentration ofarabinoxylans in plant.

[0008] The modulation of hemicellulose content can also be utilized tocontrol plant growth. For example, plant growth is determined byconcerted synthesis of cell wall polymers. It is expected that increasedsynthesis of one of the cell wall polymers, such as hemicellulose, willcause an increase in the synthesis of the rest of the polymers as well.It is expected that increased production of arabinoxylan or xyloglucanin vegetative tissue will accelerate plant growth. In contrast, it isexpected that decreased production of arabinoxylan will slow plantgrowth. Additionally, tissue-specific control of hemicelluloseproductivity is used to modify plant organ growth and development. Earlyflowering, larger fruit size, or stronger stalk or stem quality isachieved by operably linking a tissue specific promoter to a gene whichwhen expressed increases hemicellulose biosynthesis (U.S. Pat. No.6,194,638). In view of the foregoing, it would be desirable to modulatethe arabinoxylan and xyloglucan concentration in crop plants.

[0009] Clearly, modulating the concentrations of polysaccharides invarious crops is a desirable goal. However, a direct approach using theenzymes that synthesize polysaccharides has been obscured for some timedue to difficulties in isolating and cloning any of the plantpolysaccharide synthase genes. Polysaccharide synthase enzymes for thecommon polysaccharides are estimated to number in the hundreds.Recently, several cellulose-synthase genes have been identified. Thecellulose synthase genes share regions of homology that allow theidentification of novel genes that participate in polysaccharidesynthesis (Cutler et al. (1997) Current Biology 7: R108-R111).

[0010] Compositions and methodologies useful in the modulation ofpolysaccharide levels in plants are needed.

SUMMARY OF THE INVENTION

[0011] Compositions and methods for modulating plant polysaccharidesynthesis are provided. In particular, the present invention providesnucleotide sequences encoding polysaccharide synthase polypeptides. Morespecifically, the present invention provides the nucleotide sequencesset forth in SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29 or variants thereof. Also provided are amino acid sequences (SEQID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30) encodedby the nucleotide sequences of the invention, and biologically activevariants thereof.

[0012] Further compositions of the invention include expressioncassettes and vectors for expression of these novel sequences in plants.Transformed plant cells, plants, plant tissues, and seed are alsoprovided.

[0013] The invention further provides a method for modulation ofpolysaccharides, particularly hemicelluloses and pectins in plants. Themethod comprises stably incorporating into the genome of a plant anucleotide sequence encoding a polypeptide of the invention operablylinked to a promoter that drives expression of the sequence in theplant. Modification of plant polysaccharide levels alters thedigestability and nutritive value of the plant and improves thesanitation of livestock and poultry that have consumed the plant.Additionally, modification of plant polysaccharide levels alters plantgrowth and allows extraction of gums.

DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention provides compositions and methods for themodulation of polysaccharides in a plant. Compositions are nucleic acidmolecules comprising novel nucleotide sequences encoding polypeptidesthat are involved in polysaccharide synthesis, hereinafter referred toas “polysaccharide synthases.” Specifically, the present inventionprovides for isolated nucleic acid molecules comprising nucleotidesequences encoding the amino acid sequences shown in SEQ ID NOS: 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 or the nucleotidesequences encoding the cDNA insert of the plasmids deposited in abacterial host as Patent Deposit Nos. PTA-3610, PTA-3612, PTA-3611, orPTA-3613. Further provided are polypeptides having an amino acidsequence encoded by the nucleic acid molecules described herein, forexample those set forth in SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27,or 29, and fragments and variants thereof. Thesenucleotide sequences were identified in Zea mays.

[0015] Plasmids containing several of the nucleotide sequences of theinvention were deposited with the Patent Depository of the American TypeCulture Collection (ATCC), Manassas, Va., on Aug. 7, 2001 and assignedPatent Deposit Nos. PTA-3610, PTA-3612, PTA-3611, or PTA-3613. Thesedeposits will be maintained under the terms of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure. These deposits were made merely as aconvenience for those of skill in the art and are not an admission thata deposit is required under 35 U.S.C. § 112.

[0016] By “polysaccharide synthase” is intended the polypeptides of theinvention that are enzymes involved in the synthesis of polysaccharides.By “polysaccharide synthesis” or “synthesis of polysaccharide(s)” isintended any modification to a polymer of monosaccharide residuesincluding, but not limited to, xylose, glucose, arabinose, mannose, andgalactose. Such modifications include ligation or formation of any ofthe various bonds, oxidation, reduction, the addition or deletion of achemical moiety, particularly glucuronic acid, arabinose, acetyl,galactose, xylose, fucose, mannose, and rhamnose side chains, or anyother change that affects the structure or activity of the molecule,including rerouting a polysaccharide from one biosynthetic pathway toanother. While the present invention is not bound by any particularmechanism of polysaccharide synthesis, the sequences of the inventionmay synthesize polysaccharides by catalyzing glycosidic linkagesextending the polysaccharide polymer, attaching side chain residues, ormodifying the side chains of the polysaccharide. Hence polypeptideshaving polysaccharide synthase activity are characterized by the abilityto accelerate the chemical modification of a polysaccharide molecule.Rerouting a polysaccharide from one biosynthetic pathway to anotherresults in an increase or decrease in the level of anotherpolysaccharide, and hence alters polysaccharide composition of a plantcell, tissue, or organ.

[0017] The polysaccharide synthases of the invention are characterizedby their sequence similarity to previously identified enzymes that areknown to be involved in polysaccharide synthesis. Such enzymes include,for example, celA1 (Pear et al. (1996) Proc. Natl. Acad. Sci.93:12637-12642; Richmond et al. (2000) Plant Physiol. 124: 495-498),which is a cellulose synthase-like (Csl) polypeptide. The Cslpolypeptides share amino-acid-sequence homology to known cellulosesynthases. The Csl polypeptides contain a QxxRW motif, which may formthe substrate binding and catalytic sites of these enzymes (Richmond etal. (2000) Plant Physiology 124: 495-498), as well as 3-6 transmembranedomains at the carboxy-terminus and 1-2 transmembrane domains at theamino-terminus. Transmembrane domains anchor polypeptides to membranes,including, for example, the Golgi apparatus membrane. The polypeptidesresponsible for synthesis of polysaccharides other than cellulose andcallose, such as hemicelluloses and pectins, are membrane-associated (WO99/67404). In fact, polypeptides encoded by several Csl genes have beenlocalized to the Golgi apparatus and endoplasmic reticulum wheresynthesis of polysaccharides occurs (Favery et al. (2001) Genes Dev.15:79-89; Ray et al. (1976) Ber. Deutsch Bot Ges. Bd. 89:121-146 [citedin WO 99/67404]).

[0018] The novel polysaccharide synthases of the invention have severalfeatures in common with Csl polypeptides known in the art. These novelpolypeptides contain the QxxRW motif and at least 4 transmembranedomains. The nucleotide sequences of the invention (SEQ ID NOS: 1, 3, 5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29) encode polypeptides thatcontain 6 transmembrane domains. The Golgi localization of thepolypeptides encoded by the polysaccharide synthase nucleotide sequencesof the invention suggests these polysaccharide synthases are more likelyto synthesize polysaccharides such as hemicelluloses or pectins ratherthan cellulose or callose (Richmond et al. (2000) Plant Physiology 124:495-498). This CS1F class of genes is responsible for making the xylanbackbone of arabinoxylan, and in so doing provides for changes in maizestalk and other tissues. Hence, the sequences of the invention may finduse in the modulation of polysaccharide levels, thereby altering overallpolysaccharide composition of a plant cell, tissue, or organ.

[0019] Polysaccharides predominate in the cell wall of plants and aregrouped in several classes, including hemicellulose and pectin. Thehemicellulose class of polysaccharides cannot be extracted from theplant cell wall with water or chelating agents, but can be extractedwith aqueous alkali. The hemicelluloses include polysaccharides selectedfrom the group comprised of xylans, glucuronoxylans, arabinoxylans,arabinogalactans II, glucomannans, xyloglucans, mixed-link glucans, andgalactomannans. Xylans contain a backbone of (1,4)-linked xyloseresidues with side chains present in varying amounts. Inglucuronoxylans, glucuronic acid side chains predominate, although thecompound may contain arabinose and acetyl side chains also. Inarabinoxylans, arabinose side chains predominate. Glucomannans containglucose and xylose linked by 1,4-glycosidic bonds, and galactose sidechains are possible. Xyloglucans contain a backbone of (1,4)-linkedglucose residues with xylose side chains, although galactose, fucose,and arabinose side chains are possible.

[0020] The pectin class of polysaccharides can be extracted from theplant cell wall with hot aqueous solutions of chelating agents or withhot dilute acid. Pectin includes polysaccharides rich in galacturonicacid, rhamnose, arabinose, and galactose, such as polygalacturonans,rhamnogalacturonans, and some arabinans, galactans, andarabinogalactans. Polygalacturonans consist primarily of galacturonicacid. Rhamnogalacturonans consist predominantly of galacturonic acid andrhamnose, although some forms may have up to four additional types ofsugar. Galactans are polymers of galactose.

[0021] The quantity and complexity of plant polysaccharides has sloweddevelopment in the understanding of their biosynthetic pathways. Thequantity and permutations of linkages, side chain patterns, and variousbackbones in polysaccharides suggests that the number of polysaccharidesynthases is substantial. Numerous polysaccharide synthesis enzymaticactivities have been identified including, but not limited to,xyloglucan alpha,1-2 fucosyltransferase; galactinol synthase; KOJAK;sucrose:sucrose 1-fructosyltransferase; fructan:fructan1-fructosyltransferase; and Suc:fructan-6-fructosyltransferase. SeeWulff et al. (2000) Plant Physiol. 122:867-877; Sprenger et al. (2000)Plant J. 21:249-258; Favery et al. (2001) Genes Dev. 15:79-89; Reid(2000) Curr. Opin. Plant Biol. 3:512-516; Hellwege et al. (2000) Proc.Natl. Acad. Sci. 15:8699-8704; Muller et al. (2000) Plant Physiol.123:265-274; Geshi et al. (2000) Planta 210:622-629, and U.S. Pat. No.6,194,638, each of which is herein incorporated by reference.

[0022] The invention encompasses isolated or substantially purifiednucleic acid or protein compositions. An “isolated” or “purified”nucleic acid molecule or protein, or biologically active portionthereof, is substantially or essentially free from components thatnormally accompany or interact with the nucleic acid molecule or proteinas found in its naturally occurring environment. Thus, an isolated orpurified nucleic acid molecule or protein is substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. Preferably, an “isolated” nucleicacid is free of sequences (preferably protein encoding sequences) thatnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. For example, in various embodiments,the isolated nucleic acid molecule can contain less than about 5 kb, 4kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences thatnaturally flank the nucleic acid molecule in genomic DNA of the cellfrom which the nucleic acid is derived. A protein that is substantiallyfree of cellular material includes preparations of protein having lessthan about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminatingprotein. When the protein of the invention or biologically activeportion thereof is recombinantly produced, preferably culture mediumrepresents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) ofchemical precursors or non-protein-of-interest chemicals.

[0023] Fragments and variants of the disclosed nucleotide sequences andpolysaccharide synthase polypeptides encoded thereby are alsoencompassed by the present invention. By “fragment” is intended aportion of the nucleotide sequence or a portion of the amino acidsequence and hence polypeptide encoded thereby. Fragments of anucleotide sequence may encode protein fragments that retain theactivity of polysaccharide synthase polypeptides and hence function inpolysaccharide synthesis. Alternatively, fragments of a nucleotidesequence that are useful as hybridization probes generally do not encodeprotein fragments retaining the activity of polysaccharide synthases.Furthermore, fragments used to decrease the activity of a polypeptideinvolved in polysaccharide synthesis using antisense or cosuppressiontechnology also may not encode a polypeptide having the activity ofpolysaccharide synthases. However, expression of such fragments doesresult in a decrease in activity of a polypeptide involved inpolysaccharide synthesis.

[0024] Generally, fragments of a nucleotide sequence will retainbiological activity or encode a polypeptide that retains biologicalactivity wherein “biological activity” is defined as any activity orfunction of the sequences of the invention, including, but not limitedto: hybridization capability, the ability to prime synthesis, theability to be specifically labeled, cellular activity, enzymaticactivity, antigen activity, and binding activity. Thus, fragments of anucleotide sequence of the invention may range from at least about 16nucleotides, about 20 nucleotides, about 50 nucleotides, 100, 150, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500,1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, or up to 1919nucleotides for SEQ ID NO: 1; at least about 16 nucleotides, about 20nucleotides, about 50 nucleotides, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100,1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, or up to 1569nucleotides for SEQ ID NO: 3, the coding sequence set forth in SEQ IDNO: 1; at least about 16 nucleotides, about 20 nucleotides, about 50nucleotides, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300,1350, 1400, 1450, 1500, 1550, 1600, 1650, or up to 1673 nucleotides forSEQ ID NO: 5; at least about 16 nucleotides, about 20 nucleotides, about50 nucleotides, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250,1300, 1350, 1400, 1450, 1500, 1550, 1600, or up to 1611 nucleotides forSEQ ID NO: 7, the coding sequence set forth in SEQ ID NO: 5; at leastabout 16 nucleotides, about 20 nucleotides, about 50 nucleotides, 100,150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800,850, 900, 950, 1000, 1050, 1100, 1150, 1200, or up to 1221 nucleotidesfor SEQ ID NO: 9; at least about 16 nucleotides, about 20 nucleotides,about 50 nucleotides, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, or up to 1065nucleotides for SEQ ID NO: 11, the coding sequence set forth in SEQ IDNO: 9; or at least about 16 nucleotides, about 20 nucleotides, about 50nucleotides, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300,1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, or upto 1899 nucleotides for SEQ ID NO: 13; at least about 16 nucleotides,about 20 nucleotides, about 50 nucleotides, 100, 150, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000,1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, or upto 1587 nucleotides for SEQ ID NO: 15, the coding sequence set forth inSEQ ID NO: 13, at least about 16 nucleotides, about 20 nucleotides,about 50 nucleotides, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200,1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800,1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400,2450, 2500, or up to 2551 nucleotides for SEQ ID NO: 17, at least about16 nucleotides, about 20 nucleotides, about 50 nucleotides, 100, 150,200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450,1500, 1550, 1600, 1650, 1700, or up to 1740 nucleotides for SEQ ID NO:19, at least about 16 nucleotides, about 20 nucleotides, about 50nucleotides, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300,1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, or up to1834 nucleotides for SEQ ID NO: 21, at least about 16 nucleotides, about20 nucleotides, about 50 nucleotides, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100,1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700,1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300,2350, 2400, or up to 2432 nucleotides for SEQ ID NO: 23, at least about16 nucleotides, about 20 nucleotides, about 50 nucleotides, 100, 150,200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,900, 950, 1000, 1050, 1100, 1150, or up to 1190 nucleotides for SEQ IDNO: 25, at least about 16 nucleotides, about 20 nucleotides, about 50nucleotides, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300,1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900,1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300 or up to 2351 nucleotidesfor SEQ ID NO: 27, or at least about 16 nucleotides, about 20nucleotides, about 50 nucleotides, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100,1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700,1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300,or up to 2318 nucleotides for SEQ ID NO: 29. Alternatively, a nucleicacid molecule that is a fragment of a polysaccharide synthase nucleotidesequence of the present invention comprises a nucleotide sequenceconsisting of nucleotides 1-100, 100-200, 200-300, 300-400, 400-500,500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1100, 1100-1200,1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1700, 1700-1800,1800-1900, 1900-1919 of SEQ ID NO: 1; nucleotides 1-100, 100-200,200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000,1000-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500, 1500-1569 for SEQID NO: 3; nucleotides 1-100, 100-200, 200-300, 300-400, 400-500,500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1100, 1100-1200,1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1673 of SEQ ID NO: 5;nucleotides 1-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700,700-800, 800-900, 900-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400,1400-1500, 1500-1600, 1600-1611 of SEQ ID NO: 7; nucleotides 1-100,100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900,900-1000, 1000-1100, 1100-1200, 1200-1221 of SEQ ID NO: 9; nucleotides1-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800,800-900, 900-1000, 1000-1065 of SEQ ID NO: 11; nucleotides 1-100,100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900,900-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500,1500-1600, 1600-1700, 1700-1800, 1800-1899 of SEQ ID NO: 13; nucleotides1-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800,800-900, 900-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400,1400-1500, 1500-1587 of SEQ ID NO: 15, nucleotides 1-100, 100-200,200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000,1000-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500, 1500-1600,1600-1700, 1700-1800, 1800-1900, 1900-2000, 2000-2100, 2100-2200,2200-2300, 2300-2400, 2400-2500, 2500-2551 of SEQ ID NO: 17, nucleotides1-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800,800-900, 900-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400,1400-1500, 1500-1600, 1600-1700, 1700-1740 of SEQ ID NO: 19, nucleotides1-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800,800-900, 900-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400,1400-1500, 1500- 1600, 1600-1700, 1700-1800, 1800-1834 of SEQ ID NO: 21,nucleotides 1-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700,700-800, 800-900, 900-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400,1400-1500, 1500-1600, 1600-1700, 1700-1800, 1800-1900, 1900-2000,2000-2100, 2100-2200, 2200-2300, 2300-2400, 2400-2432 of SEQ ID NO: 23,nucleotides 1-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700,700-800, 800-900, 900-1000, 1000-1100, 1100-1190 of SEQ ID NO: 25,nucleotides 1-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700,700-800, 800-900, 900-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400,1400-1500, 1500-1600, 1600-1700, 1700-1800, 1800-1900, 1900-2000,2000-2100, 2100-2200, 2200-2300, 2300-2351 of SEQ ID NO: 27, ornucleotides 1-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700,700-800, 800-900, 900-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400,1400-1500, 1500-1600, 1600-1700, 1700-1800, 1800-1900, 1900-2000,2000-2100, 2100-2200, 2200-2300, 2300-2318 of SEQ ID NO: 29.

[0025] A fragment of a polysaccharide synthase nucleotide sequence thatencodes a biologically active portion of a polysaccharide synthasepolypeptide of the invention will encode at least 15, 25, 30, 50, 100,150, 200, 250, 300, 350, 400, 450, 500, or up to 522 contiguous aminoacids present in SEQ ID NO: 2 or SEQ ID NO: 4; at least 15, 25, 30, 50,100, 150, 200, 250, 300, 350, 400, 450, 500, or up to 536 contiguousamino acids present in SEQ ID NO: 6 or SEQ ID NO: 8; at least 15, 25,30, 50, 100, 150, 200, 250, 300, 350, or up to 354 contiguous aminoacids present in SEQ ID NO: 10 or SEQ ID NO: 12; at least 15, 25, 30,50, 100, 150, 200, 250, 300, 350, 400, 450, 500,or up to 528 contiguousamino acids present in SEQ ID NO: 14 or SEQ ID NO: 16, at least 15, 25,30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,or up to 720 contiguous amino acids present in SEQ ID NO: 18, at least15, 25, 30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, or up to537 contiguous amino acids present in SEQ ID NO: 20, at least 15, 25,30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550 or up to 572contiguous amino acids present in SEQ ID NO: 22, at least 15, 25, 30,50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, orup to 727 contiguous amino acids present in SEQ ID NO: 24, at least 15,25, 30, 50, 100, 150, 200, 250,or up to 264 contiguous amino acidspresent in SEQ ID NO: 26, at least 15, 25, 30, 50, 100, 150, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, or up to 741 contiguousamino acids present in SEQ ID NO: 28, at least 15, 25, 30, 50, 100, 150,200, 250, 300, 350, 400, 450, 500, 550 or up to 590 contiguous aminoacids present in SEQ ID NO: 30. Fragments of a polysaccharide synthasenucleotide sequence that are useful as hybridization probes or PCRprimers generally need not encode a polypeptide that retainspolysaccharide synthase activity.

[0026] Thus, a fragment of a polysaccharide synthase nucleotide sequencemay encode a biologically active portion of a polysaccharide synthasepolypeptide, or it may be a fragment that can be used as a hybridizationprobe or PCR primer using methods disclosed below. A biologically activeportion of a polysaccharide synthase polypeptide can be prepared byisolating a portion of one of the nucleotide sequences of the invention,expressing the encoded portion of the polysaccharide synthasepolypeptide (e.g., by recombinant expression in vitro), and assessingthe activity of the encoded portion of the polysaccharide synthasepolypeptide.

[0027] Variants of the novel nucleotide sequences or polypacchaidesynthase polypeptides encoded thereby are also encompassed by thepresent invention. By “variants” is intended substantially similarsequences. For nucleotide sequences, conservative variants include thosesequences that, because of the degeneracy of the genetic code, encodethe amino acid sequence of one of the polysaccharide synthasepolypeptides of the invention. Naturally occurring allelic variants suchas these can be identified with the use of well-known molecular biologytechniques, as, for example, with polymerase chain reaction (PCR) andhybridization techniques as outlined below. Variant nucleotide sequencesalso include synthetically derived nucleotide sequences, such as thosegenerated, for example, by using site-directed mutagenesis but whichstill encode a polysaccharide synthase polypeptide of the invention.Generally, variants of a particular nucleotide sequence of the inventionwill have at least about 65%, 70%, generally at least about 75%, 80%,85%, preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,and more preferably at least about 98%, 99% or more sequence identity tothat particular nucleotide sequence as determined by sequence alignmentprograms described elsewhere herein using default parameters.

[0028] By “variant” protein is intended a protein derived from thenative protein by deletion (so-called truncation) or addition of one ormore amino acids to the N-terminal and/or C-terminal end of the nativeprotein; deletion or addition of one or more amino acids at one or moresites in the native protein; or substitution of one or more amino acidsat one or more sites in the native protein. Variant proteins encompassedby the present invention are biologically active, that is they continueto possess a desired biological activity of the native protein,particularly, polysaccharide synthesis activity as described herein.Such variants may result from, for example, genetic polymorphism or fromhuman manipulation. Biologically active variants of a nativepolysaccharide synthase protein of the invention will have at leastabout 50%, 60%, 65%, 70%, generally at least about 75%, 80%, 85%,preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, andmore preferably at least about 98%, 99% or more sequence identity to theamino acid sequence for the native protein as determined by sequencealignment programs described elsewhere herein using default parameters.A biologically active variant of a protein of the invention may differfrom that protein by as few as 1-15 amino acid residues, as few as 1-10,such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acidresidue.

[0029] The proteins of the invention may be altered in various waysincluding amino acid substitutions, deletions, truncations, andinsertions. Methods for such manipulations are generally known in theart. For example, amino acid sequence variants of the polysaccharidesynthase polypeptides can be prepared by mutations in the DNA. Methodsfor mutagenesis and nucleotide sequence alterations are well known inthe art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S.Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques inMolecular Biology (MacMillan Publishing Company, New York) and thereferences cited therein. Guidance as to appropriate amino acidsubstitutions that do not affect biological activity of the protein ofinterest may be found in the model of Dayhoff et al. (1978) Atlas ofProtein Sequence and Structure (Natl. Biomed. Res. Found., Washington,D.C.), herein incorporated by reference. Conservative substitutions,such as exchanging one amino acid with another having similarproperties, may be preferable.

[0030] Thus, the genes and nucleotide sequences of the invention includeboth the naturally occurring sequences as well as mutant forms.Likewise, the proteins of the invention encompass both naturallyoccurring proteins as well as variations and modified forms thereof.Such variants will continue to possess the desired polysaccharidesynthase activity. Obviously, the mutations that will be made in the DNAencoding the variant must not place the sequence out of reading frameand preferably will not create complementary regions that could producesecondary mRNA structure. See EP Patent Application Publication No.75,444.

[0031] The deletions, insertions, and substitutions of the proteinsequences encompassed herein are not expected to produce radical changesin the characteristics of the protein. However, when it is difficult topredict the exact effect of the substitution, deletion, or insertion inadvance of doing so, one skilled in the art will appreciate that theeffect will be evaluated by routine screening assays. That is, theactivity can be evaluated by UDP-substrate binding studies, including atest battery assay to determine optimal substrates. See, for example,Pear et a. (1996) Proc. Natl. Acad. Sci. 93:12637-12642; Geshi et al.(2000) Planta 210:622-629; Wulff et al. (2000) Plant Physiol.122:867-877) herein incorporated by reference.

[0032] Variant nucleotide sequences and proteins also encompasssequences and proteins derived from a mutagenic and recombinogenicprocedure such as DNA shuffling. With such a procedure, one or moredifferent polysaccharide synthase coding sequences can be manipulated tocreate a new polysaccharide synthase possessing the desired properties.In this manner, libraries of recombinant polynucleotides are generatedfrom a population of related sequence polynucleotides comprisingsequence regions that have substantial sequence identity and can behomologously recombined in vitro or in vivo. For example, using thisapproach, sequence motifs encoding a domain of interest may be shuffledbetween a polysaccharide synthase sequences of the invention and otherknown polysaccharide synthase genes to obtain a new gene coding for apolysacchaide synthase with an improved property of interest, such as anincreased K_(m). Strategies for such DNA shuffling are known in the art.See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997)Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol.272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat.Nos. 5,605,793 and 5,837,458.

[0033] The nucleotide sequences of the invention can be used to isolatecorresponding sequences from other organisms, particularly other plants,and more particularly other monocots. In this manner, methods such asPCR, hybridization, and the like can be used to identify such sequencesbased on their sequence homology to the sequences set forth herein.Sequences isolated based on their sequence identity to the entirepolysaccharide synthase sequences set forth herein or to fragmentsthereof are encompassed by the present invention. Such sequences includesequences that are orthologs of the disclosed sequences. By “orthologs”is intended genes derived from a common ancestral gene and which arefound in different species as a result of speciation. Genes found indifferent species are considered orthologs when their nucleotidesequences and/or their encoded protein sequences share substantialidentity as defined elsewhere herein. Functions of orthologs are oftenhighly conserved among species.

[0034] In a PCR approach, oligonucleotide primers can be designed foruse in PCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any plant of interest. Methods for designingPCR primers and PCR cloning are generally known in the art and aredisclosed in Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods andApplications (Academic Press, New York); Innis and Gelfand, eds. (1995)PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds.(1999) PCR Methods Manual (Academic Press, New York). Known methods ofPCR include, but are not limited to, methods using paired primers,nested primers, single specific primers, degenerate primers,gene-specific primers, vector-specific primers, partially-mismatchedprimers, and the like.

[0035] In hybridization techniques, all or part of a known nucleotidesequence is used as a probe that selectively hybridizes to othercorresponding nucleotide sequences present in a population of clonedgenomic DNA fragments or cDNA fragments (i.e., genomic or cDNAlibraries) from a chosen organism. The hybridization probes may begenomic DNA fragments, cDNA fragments, RNA fragments, or otheroligonucleotides, and may be labeled with a detectable group such as³²P, or any other detectable marker. Thus, for example, probes forhybridization can be made by labeling synthetic oligonucleotides basedon the polysaccharide synthase sequences of the invention. Methods forpreparation of probes for hybridization and for construction of cDNA andgenomic libraries are generally known in the art and are disclosed inSambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed.,Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

[0036] For example, the entire polysaccharide synthase sequencesdisclosed herein, or one or more portions thereof, may be used as aprobe capable of specifically hybridizing to correspondingpolysaccharide synthase sequences and messenger RNAs. To achievespecific hybridization under a variety of conditions, such probesinclude sequences that are unique among polysaccharide synthasesequences and are preferably at least about 10 nucleotides in length,and most preferably at least about 20 nucleotides in length. Such probesmay be used to amplify corresponding polysaccharide synthase sequencesfrom a chosen plant by PCR. This technique may be used to isolateadditional coding sequences from a desired plant or as a diagnosticassay to determine the presence of coding sequences in a desired plant.Hybridization techniques include hybridization screening of plated DNAlibraries (either plaques or colonies; see, for example, Sambrook et al.(1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold SpringHarbor Laboratory Press, Plainview, N.Y.).

[0037] Hybridization of such sequences may be carried out understringent conditions. By “stringent conditions” or “stringenthybridization conditions” is intended conditions under which a probewill hybridize to its target sequence to a detectably greater degreethan to other sequences (e.g., at least 2-fold over background).Stringent conditions are sequence-dependent and will be different indifferent circumstances. By controlling the stringency of thehybridization and/or washing conditions, target sequences that are 100%complementary to the probe can be identified (homologous probing).Alternatively, stringency conditions can be adjusted to allow somemismatching in sequences so that lower degrees of similarity aredetected (heterologous probing). Generally, a probe is less than about1000 nucleotides in length, preferably less than 500 nucleotides inlength.

[0038] Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl,1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffersmay comprise about 0.1% to about 1% SDS. Duration of hybridization isgenerally less than about 24 hours, usually about 4 to about 12 hours.

[0039] Specificity is typically the function of post-hybridizationwashes, the critical factors being the ionic strength and temperature ofthe final wash solution. For DNA-DNA hybrids, the T_(m) can beapproximated from the equation of Meinkoth and Wahl (1984) Anal.Biochem. 138:267-284: T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (%form)−500/L; where M is the molarity of monovalent cations, % GC is thepercentage of guanosine and cytosine nucleotides in the DNA, % form isthe percentage of formamide in the hybridization solution, and L is thelength of the hybrid in base pairs. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of a complementary targetsequence hybridizes to a perfectly matched probe. T_(m) is reduced byabout 1° C. for each 1% of mismatching; thus, T_(m), hybridization,and/or wash conditions can be adjusted to hybridize to sequences of thedesired identity. For example, if sequences with ≧90% identity aresought, the T_(m) can be decreased 10° C. Generally, stringentconditions are selected to be about 5° C. lower than the thermal meltingpoint (T_(m)) for the specific sequence and its complement at a definedionic strength and pH. However, severely stringent conditions canutilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower than thethermal melting point (T_(m)); moderately stringent conditions canutilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower thanthe thermal melting point (T_(m)); low stringency conditions can utilizea hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution), it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen (1993)Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part 1, Chapter 2(Elsevier, N.Y.); and Ausubel et al., eds. (1995) Current Protocols inMolecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience,New York). See Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

[0040] Thus, isolated sequences that encode for a polysaccharidesynthase protein and which hybridize under stringent conditions to thepolysaccharide synthase sequences disclosed herein, or to fragmentsthereof, are encompassed by the present invention.

[0041] The following terms are used to describe the sequencerelationships between two or more nucleic acids or polynucleotides: (a)“reference sequence”, (b) “comparison window”, (c) “sequence identity”,(d) “percentage of sequence identity”, and (e) “substantial identity”.

[0042] (a) As used herein, “reference sequence” is a defined sequenceused as a basis for sequence comparison. A reference sequence may be asubset or the entirety of a specified sequence; for example, as asegment of a full-length cDNA or gene sequence, or the complete cDNA orgene sequence.

[0043] (b) As used herein, “comparison window” makes reference to acontiguous and specified segment of a polynucleotide sequence, whereinthe polynucleotide sequence in the comparison window may compriseadditions or deletions (i.e., gaps) compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. Generally, the comparison window is at least 20contiguous nucleotides in length, and optionally can be 30, 40, 50, 100,or longer. Those of skill in the art understand that to avoid a highsimilarity to a reference sequence due to inclusion of gaps in thepolynucleotide sequence a gap penalty is typically introduced and issubtracted from the number of matches.

[0044] Methods of alignment of sequences for comparison are well knownin the art. Thus, the determination of percent sequence identity betweenany two sequences can be accomplished using a mathematical algorithm.Preferred, non-limiting examples of such mathematical algorithms are thealgorithm of Myers and Miller (1988) CABIOS 4:11-17; the local homologyalgorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the homologyalignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol.48:443-453; the search-for-similarity-method of Pearson and Lipman(1988) Proc. Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin andAltschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as inKarlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

[0045] Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Version 8 (availablefrom Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.(1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153;Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992)CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.The ALIGN program is based on the algorithm of Myers and Miller (1988)supra. A PAM120 weight residue table, a gap length penalty of 12, and agap penalty of 4 can be used with the ALIGN program when comparing aminoacid sequences. The BLAST programs of Altschul et al (1990) J. Mol.Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990)supra. BLAST nucleotide searches can be performed with the BLASTNprogram, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to a nucleotide sequence encoding a protein of the invention.BLAST protein searches can be performed with the BLASTX program,score=50, wordlength=3, to obtain amino acid sequences homologous to aprotein or polypeptide of the invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389.Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform aniterated search that detects distant relationships between molecules.See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST,PSI-BLAST, the default parameters of the respective programs (e.g.,BLASTN for nucleotide sequences, BLASTX for proteins) can be used. Seehttp://www.ncbi.nlm.nih.gov. Alignment may also be performed manually byinspection.

[0046] Unless otherwise stated, sequence identity/similarity valuesprovided herein refer to the value obtained using GAP version 10 usingthe following parameters: % identity using GAP Weight of 50 and LengthWeight of 3; % similarity using Gap Weight of 12 and Length Weight of 4,or any equivalent program. By “equivalent program” is intended anysequence comparison program that, for any two sequences in question,generates an alignment having identical nucleotide or amino acid residuematches and an identical percent sequence identity when compared to thecorresponding alignment generated by GAP Version 10.

[0047] GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol.Biol. 48: 443-453, to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. Default gap creation penalty values and gapextension penalty values in Version 10 of the Wisconsin GeneticsSoftware Package for protein sequences are 8 and 2, respectively. Fornucleotide sequences the default gap creation penalty is 50 while thedefault gap extension penalty is 3. The gap creation and gap extensionpenalties can be expressed as an integer selected from the group ofintegers consisting of from 0 to 200. Thus, for example, the gapcreation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

[0048] GAP presents one member of the family of best alignments. Theremay be many members of this family, but no other member has a betterquality. GAP displays four figures of merit for alignments: Quality,Ratio, Identity, and Similarity. The Quality is the metric maximized inorder to align the sequences. Ratio is the quality divided by the numberof bases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

[0049] (c) As used herein, “sequence identity” or “identity” in thecontext of two nucleic acid or polypeptide sequences makes reference tothe residues in the two sequences that are the same when aligned formaximum correspondence over a specified comparison window. Whenpercentage of sequence identity is used in reference to proteins it isrecognized that residue positions which are not identical often differby conservative amino acid substitutions, where amino acid residues aresubstituted for other amino acid residues with similar chemicalproperties (e.g., charge or hydrophobicity) and therefore do not changethe functional properties of the molecule. When sequences differ inconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Sequences that differ by such conservative substitutionsare said to have “sequence similarity” or “similarity”. Means for makingthis adjustment are well known to those of skill in the art. Typicallythis involves scoring a conservative substitution as a partial ratherthan a full mismatch, thereby increasing the percentage sequenceidentity. Thus, for example, where an identical amino acid is given ascore of 1 and a non-conservative substitution is given a score of zero,a conservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif.).

[0050] (d) As used herein, “percentage of sequence identity” means thevalue determined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

[0051] (e)(i) The term ”substantial identity” of polynucleotidesequences means that a polynucleotide comprises a sequence that has atleast 70% sequence identity, preferably at least 80%, more preferably atleast 90%, and most preferably at least 95%, compared to a referencesequence using one of the alignment programs described using standardparameters. One of skill in the art will recognize that these values canbe appropriately adjusted to determine corresponding identity ofproteins encoded by two nucleotide sequences by taking into accountcodon degeneracy, amino acid similarity, reading frame positioning, andthe like. Substantial identity of amino acid sequences for thesepurposes normally means sequence identity of at least 60%, morepreferably at least 70%, 80%, 90%, and most preferably at least 95%.

[0052] Another indication that nucleotide sequences are substantiallyidentical is if two molecules hybridize to each other under stringentconditions. Generally, stringent conditions are selected to be about 5°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. However, stringentconditions encompass temperatures in the range of about 1° C. to about20° C. lower than the T_(m), depending upon the desired degree ofstringency as otherwise qualified herein. Nucleic acids that do nothybridize to each other under stringent conditions are stillsubstantially identical if the polypeptides they encode aresubstantially identical. This may occur, e.g., when a copy of a nucleicacid is created using the maximum codon degeneracy permitted by thegenetic code. One indication that two nucleic acid sequences aresubstantially identical is when the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid.

[0053] (e)(ii) The term “substantial identity” in the context of apeptide indicates that a peptide comprises a sequence with at least 70%sequence identity to a reference sequence, preferably 80%, morepreferably 85%, most preferably at least 90% or 95% sequence identity tothe reference sequence over a specified comparison window. Preferably,optimal alignment is conducted using the homology alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48:443-453. An indication thattwo peptide sequences are substantially identical is that one peptide isimmunologically reactive with antibodies raised against the secondpeptide. Thus, a peptide is substantially identical to a second peptide,for example, where the two peptides differ only by a conservativesubstitution. Peptides that are “substantially similar” share sequencesas noted above except that residue positions that are not identical maydiffer by conservative amino acid changes.

[0054] Methods are provided for modulating polysaccharide synthaselevels in a plant. By “modulating” is intended decreasing or increasingthe native levels of polysaccharide synthase transcripts, polypeptides,enzyme activity; altering the enzyme specificity; or a combinationthereof. By “decreasing” polysaccharide synthase transcripts,polypeptides, or enzyme activity is intended a 1%, 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 100% reduction of the native polysaccharide transcript, polypeptideor enzyme activity. By “increasing” polysaccharide synthase transcripts,polypeptides, or enzyme activity is intended a 1%, 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 100% or more augmentation of the native polysaccharide transcript,polypeptide, or enzyme activity. Modulating also comprises expression ofan enzyme normally not found in a particular plant. Thus, plants andplant cells are obtained that have altered polysaccharide biosynthesispathways. Such plants, plant cells, and plant tissues are “modified” inthat the activities of proteins in polysaccharide biosynthesis pathwaysare altered. As noted below, various methods are available for creatingmodified plants, plant cells, and plant tissues, includingtransformation, transcription, and breeding. Any method known in the artfor modulating expression may be employed singly or in combination toachieve the desired result. Such techniques will lead to an alteredexpression of polysaccharide synthase polypeptides involved in thepolysaccharide biosynthesis pathways in the modified plant, plant cell,or plant tissue.

[0055] Modulating can be accomplished by either up-regulating ordown-regulating expression of a nucleotide sequence of the invention. Anembodiment of the invention involves modulation of polysaccharidesynthase expression in a crop plant, particularly maize. Methods forup-regulating expression of a nucleotide sequence include introducing anucleotide sequence of the invention operably linked to a heterologouspromoter such as a strong promoter, constitutive promoter, orseed-specific promoter into a plant cell of interest. Methods fordown-regulating expression of a nucleotide sequence include the use ofantisense suppression and co-suppression technology to inhibitexpression of a nucleotide sequence of the invention.

[0056] Anti-sense suppression technology is a method of down-regulatingexpression of the nucleotide sequences of the invention. It isrecognized that with these nucleotide sequences, antisense constructionscomplementary to at least a portion of the messenger RNA (mRNA) for thepolysaccharide synthase sequences can be constructed. Antisensenucleotides are constructed to hybridize with the corresponding mRNA.Modifications of the antisense sequences may be made as long as thesequences hybridize to and interfere with expression of thecorresponding mRNA. In this manner, antisense constructions having 70%,preferably 80%, more preferably 85% sequence identity to thecorresponding antisense sequences may be used. Furthermore, portions ofthe antisense nucleotides may be used to disrupt the expression of thetarget sequence. Generally, sequences of at least 50 nucleotides, 100nucleotides, 200 nucleotides, or greater may be used.

[0057] The nucleotide sequences of the present invention may also beused in the sense orientation to suppress the expression of endogenouspolysaccharide synthases in plants. Methods for suppressing geneexpression in plants using nucleotide sequences in the sense orientationare known in the art. The methods generally involve transforming plantswith a DNA construct comprising a promoter that drives expression in aplant operably linked to at least a portion of a nucleotide sequencethat corresponds to the transcript of the endogenous gene. Typically,such a nucleotide sequence has substantial sequence identity to thesequence of the transcript of the endogenous gene, preferably greaterthan about 65% sequence identity, more preferably greater than about 85%sequence identity, most preferably greater than about 95% sequenceidentity. See U.S. Pat. Nos. 5,283,184 and 5,034,323; hereinincorporated by reference.

[0058] Alternatively, polysaccharide synthase expression may bemodulated by modifying the kinetic properties of an endogenouspolysaccharide synthase through site-directed alterations of the codingsequence of the endogenous gene resulting in changes in the amino acidsequence of the encoded enzyme. Such site-directed alterations may beaccomplished by any method known in the art including, but not limitedto, a chimeraplasty-based method involving a nucleotide construct of theinvention.

[0059] In one embodiment of the invention a method for improving thedigestibility of grain crops is provided. By “digestibility” is intendedthe percentage of a substance taken into a digestive tract that isabsorbed by the body. Arabinoxylans constitute 45%-65% of the grain cellwall, but they impede digestion of the grain and may sequesterdigestible components of grain thus reducing digestibility (WO 99/67404;van der Klis et al. (1995) Anim. Feed Sci. & Tech. 51:15-27). The highlevels of undigestible material contribute to the sanitation challengesof livestock and poultry raising (Selinger et al. (1996) Anaerobe2:263-284). The methods for modulating polysaccharide synthase levelscan be used to increase digestibility of grain and forage crops bylowering the concentration of polysaccharide synthases, thereby loweringthe concentration of hemicelluloses, such as arabinoxylan, in themodified plant. Tissue-specific promoters can be used to direct downregulation of expression of the nucleotide sequences of the invention inthe desired plant tissues using antisense or sense-suppressiontechnology as described elsewhere herein.

[0060] Methods to measure digestibility are known in the art andinclude, but are not limited to, determining the food conversion ratio(WO 99/67404), sampling chyme for chromium, phosphorous, calcium,magnesium, sodium, and potassium (van der Klis et al. (1995) Anim. FeedSci. & Tech. 51:15-27), in sacco degradation (van Vuuren et al. (1989)Grass & Forage Sci. 44: 223-230), growth studies (GrootWassink et al.(1989) J. Sci. Food Agric. 46:289-300), and the enzyme digestible drymatter (EDDM) assay (Boisen and Fernandez (1997) Animal Feed Sci. Tech.68:83-92; and Boisen and Fernandez (1995) Animal Feed Sci. Tech.51:29-43); all of which are herein incorporated by reference. Suchmethods can be used to determine the digestibility and/or energyavailability of the plant parts of plants modified in accordance withmethods of the invention. The modified plant parts, such as modifiedgrain, may be fed to a variety of livestock including, but not limitedto, poultry, cattle, swine, horses, and sheep.

[0061] In another embodiment of the invention a method for improving gumextractability is provided. By “gum” is intended any of numerouscolloidal polysaccharides of plant origin that are gelatinous when moistbut which harden on drying, including, but not limited to,arabinoxylans, galactans, and mixed-link glucans. Whereas high gumconcentration can be detrimental to digestibility, there is a stronginterest in their industrial applications, such as their use asthickeners in the food industry (Sanderson (1982) Prog. Fd. Nutr. Sci.6:77-87). About 15% of the total corn produced in the USA is subjectedto wet milling to produce mainly starch and also oil from the germ. Wetmilling is a multi-step process involving the steeping and grinding ofkernels, and separating the kernels into starch, protein, oil, and fiberportions. See S. R. Eckhoff (1992) Proceedings of the 4^(th) CornUtilization Conference, Jun. 24-26, 1992, St. Louis, Mo., (National CornGrowers Association, CIBA-GEIGY Seed Division, and the USDA). The fiberresidue left at the end of the wet-milling process is rich inarabinoxylans. However, it is not currently economically feasible toextract arabinoxylans from the wet-milled residue of corn. Increasingthe level of arabinoxylans, galactans, or mixed-link glucans in themaize grain improves the ability to extract the gums. This can beachieved by generating a plant that overexpresses polysaccharidesynthases involved in synthesis of arabinoxylans, galactans, andmixed-link glucans, particularly overexpression in the tissue ofinterest, such as grain.

[0062] The present invention also provides a method for modulating theplant growth rate. Plant cell growth is accomplished through looseningof the plant cell wall and expansion due to the turgor pressure of theplant cell. There is a relationship between the looseness of the plantcell wall and the turgor pressure of the cell such that looser cellwalls require less turgor pressure to expand, while stronger cell wallsrequire more turgor pressure to expand. A component of cell wallloosening is the deposition by a process known as intussusception ofmatrix polysaccharides within the cell wall. The newly incorporatedpolysaccharides relieve stress in the load-bearing components of theplant cell wall and prevent a perpetual gradual thinning of the cellwalls during plant cell growth. Under conditions of drought or stress,the turgor pressure of the cell decreases, and the plant decreasessynthesis of the polysaccharides necessary for cell-wall loosening andcell growth (see Ray (1992) Curr. Topics in Plant Biochem. & Phys.11:18-41). In this manner, the interplay between low turgor pressure andthe strength of the cell wall prevents or slows growth. Increasedsynthesis of polysaccharides would allow the plant cell wall to loosenand allow growth with less turgor pressure. The use of stress-responsivepromoters would allow regulated expression of the polysaccharidesynthases of the invention (see U.S. Pat. Nos: US 5,891,859; U.S. Pat.No. 5,929,305; U.S. Pat. No. 5, 965,705; U.S. Pat. No. 5,892, 009).Polysaccharide synthases of the Csl family of gene products have beenshown to be involved in plant growth (Favery et al. (2001) Genes Dev.15:79-89). Therefore, plant cell growth may be modulated by modulatingthe levels of polysaccharides through modulation of polysaccharidesynthase expression. In this manner, the nucleotide sequences of theinvention may be used to modulate the levels of polysaccharide synthesisactivity and thus to mediate plant growth.

[0063] Although modulated growth of the entire plant is one possibledesired embodiment, it is recognized that modulated growth of specifictissues such as the roots or seeds may be desired. Methods oftissue-preferred expression of the nucleotide sequences of the inventionare discussed elsewhere herein.

[0064] The polysaccharide synthase sequences of the invention areprovided in expression cassettes for expression in the plant ofinterest. The cassette will include 5′ and 3′ regulatory sequencesoperably linked to a nucleotide sequence of the invention. By “operablylinked” is intended a functional linkage between a promoter and a secondsequence, wherein the promoter sequence initiates and mediatestranscription of the DNA sequence corresponding to the second sequence.Generally, operably linked means that the nucleic acid sequences beinglinked are contiguous and, where necessary to join two protein codingregions, contiguous and in the same reading frame. The cassette mayadditionally contain at least one additional gene to be cotransformedinto the organism. Alternatively, the additional gene(s) can be providedon multiple expression cassettes.

[0065] Such an expression cassette is provided with a plurality ofrestriction sites for insertion of the polysaccharide synthase sequenceto be under the transcriptional regulation of the regulatory regions.The expression cassette may additionally contain selectable markergenes.

[0066] The expression cassette will include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region, anucleotide sequence of the invention, and a transcriptional andtranslational termination region functional in plants. Thetranscriptional initiation region, the promoter, may be native oranalogous or foreign or heterologous to the plant host. Additionally,the promoter may be the natural sequence or alternatively a syntheticsequence. By “foreign” is intended that the transcriptional initiationregion be not found in the native plant into which the transcriptionalinitiation region is introduced. As used herein, a chimeric genecomprises a coding sequence operably linked to a transcriptioninitiation region that is heterologous to the coding sequence.

[0067] While it may be preferable to express the sequences usingheterologous promoters, the native promoter sequences may be used. Suchconstructs would change expression levels of polysaccharide synthases inthe plant or plant cell. Thus, the phenotype of the plant or plant cellis altered.

[0068] The termination region may be native with the transcriptionalinitiation region, may be native with the operably linked polysaccharidesynthase sequence of interest, or may be derived from another source.Convenient termination regions are available from the Ti-plasmid of A.tumefaciens, such as the octopine synthase and nopaline synthasetermination regions. See also Guerineau et al. (1991) Mol. Gen. Genet.262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991)Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroeet al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res.17:7891-7903; and Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

[0069] Where appropriate, the polysaccharide synthase sequences may beoptimized for increased expression in the transformed plant. That is,the sequences can be synthesized using plant-preferred codons forimproved expression. See, for example, Campbell and Gowri (1990) PlantPhysiol. 92:1-11 for a discussion of host-preferred codon usage. Methodsare available in the art for synthesizing plant-preferred sequences.See, for example, U.S. Pat. Nos. 5,380,831, and 5,436,391, and Murray etal. (1989) Nucleic Acids Res. 17:477-498, herein incorporated byreference.

[0070] Additional sequence modifications are known to enhance geneexpression in a cellular host. These include elimination of sequencesencoding spurious polyadenylation signals, exon-intron splice sitesignals, transposon-like repeats, and other such well-characterizedsequences that may be deleterious to gene expression. The G-C content ofthe sequence may be adjusted to levels average for a given cellularhost, as calculated by reference to known genes expressed in the hostcell. When possible, the sequence is modified to avoid predicted hairpinsecondary mRNA structures.

[0071] The expression cassettes may additionally contain 5′ leadersequences in the expression cassette construct. Such leader sequencescan act to enhance translation. Translation leaders are known in the artand include: picornavirus leaders, for example, EMCV leader(Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al. (1989)Proc Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders, for example,TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus) (Virology154:9-20), and human immunoglobulin heavy-chain binding protein (BiP)(Macejak et al. (1991) Nature 353:90-94); untranslated leader from thecoat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al.(1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie etal. (1989) in Molecular Biology of RNA, ed. Cech (Liss, N.Y.), pp.237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et al.(1991) Virology 81:382-385). See also, Della-Cioppa et al. (1987) PlantPhysiol. 84:965-968. Other methods known to enhance translation can alsobe utilized, for example, introns, and the like.

[0072] In preparing the expression cassette, the various DNA fragmentsmay be manipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

[0073] Generally, the expression cassette will comprise a selectablemarker gene for the selection of transformed cells. Selectable markergenes are utilized for the selection of transformed cells or tissues.Marker genes include genes encoding antibiotic resistance, such as thoseencoding neomycin phosphotransferase II (NEO) and hygromycinphosphotransferase (HPT), as well as genes conferring resistance toherbicidal compounds, such as glufosinate ammonium, bromoxynil,imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). See generally,Yarranton (1992) Curr. Opin. Biotech. 3:506-511; Christopherson et al.(1992) Proc. Natl. Acad. Sci. USA 89:6314-6318; Yao et al. (1992) Cell71:63-72; Reznikoff (1992) Mol. Microbiol. 6:2419-2422; Barkley et al.(1980) in The Operon, pp. 177-220; Hu et al. (1987) Cell48:555-566;Brown et al. (1987) Cell 49:603-612; Figge et al. (1988) Cell52:713-722; Deuschle et al. (1989) Proc. Natl. Acad. Sci. USA86:5400-5404; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA86:2549-2553; Deuschle et al. (1990) Science 248:480-483; Gossen (1993)Ph.D. Thesis, University of Heidelberg; Reines et al. (1993) Proc. Natl.Acad. Sci. USA 90:1917-1921; Labow et al. (1990) Mol. Cell. Biol.10:3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA89:3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci. USA88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653;Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolbet al. (1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidtet al. (1988) Biochemistry 27:1094-1104; Bonin (1993) Ph.D. Thesis,University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci.USA 89:5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother.36:913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology,Vol. 78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature334:721-724. Such disclosures are herein incorporated by reference. Theabove list of selectable marker genes is not meant to be limiting. Anyselectable marker gene can be used in the present invention.

[0074] The use of the term “nucleotide constructs” herein is notintended to limit the present invention to nucleotide constructscomprising DNA. Those of ordinary skill in the art will recognize thatnucleotide constructs, particularly polynucleotides andoligonucleotides, comprised of ribonucleotides and combinations ofribonucleotides and deoxyribonucleotides may also be employed in themethods disclosed herein. Thus, the nucleotide constructs of the presentinvention encompass all nucleotide constructs that can be employed inthe methods of the present invention for transforming plants including,but not limited to, those comprised of deoxyribonucleotides,ribonucleotides, and combinations thereof. Such deoxyribonucleotides andribonucleotides include both naturally occurring molecules and syntheticanalogues. The nucleotide constructs of the invention also encompass allforms of nucleotide constructs including, but not limited to,single-stranded forms, double-stranded forms, hairpins, stem-and-loopstructures, and the like.

[0075] In certain embodiments the nucleic acid sequences of the presentinvention can be stacked with any combination of polynucleotidesequences of interest in order to create plants with a desiredphenotype. For example, the polynucleotides of the present invention maybe stacked with any other polynucleotides of the present invention, suchas any combination of polysaccharide synthases (SEQ ID NOS: 1, 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29), or with other genesimplicated in polysaccharide synthase enzymatic activities including,but not limited to, xyloglucan alpha 1-2 fucosyltransferase; galactinolsynthase; KOJAK; sucrose:sucrose 1-fructosyltransferase; fructan:fructan1-fructosyltransferase; and Suc:fructan-6-fructosyltransferase. (SeeWulff et al. (2000) Plant Physiol. 122:867-877; Sprenger et al. (2000)Plant J. 21:249-258; Favery et al. (2001) Genes Dev. 15:79-89; Reid(2000) Curr. Opin. Plant Biol. 3:512-516; Hellwege et al. (2000) Proc.Natl. Acad. Sci. 15:8699-8704; Muller et al. (2000) Plant Physiol.123:265-274; Geshi et al. (2000) Planta 210:622-629, and U.S. Pat. No.6,194,638, each of which is herein incorporated by reference.) Thecombinations generated can also include multiple copies of any one ofthe polynucleotides of interest. The polynucleotides of the presentinvention can also be stacked with any other gene or combination ofgenes to produce plants with a variety of desired trait combinationsincluding but not limited to traits desirable for animal feed such ashigh oil genes (e.g., U.S. Pat. No. 6,232,529); balanced amino acids(e.g. hordothionins (U.S. Pat. Nos. 5,990,389; 5,885,801; 5,885,802; and5,703,409); barley high lysine (Williamson et al. (1987) Eur. J Biochem.165:99-106; and WO 98/20122); and high methionine proteins (Pedersen etal. (1986) J. Biol. Chem. 261:6279; Kirihara et al. (1988) Gene 71:359;and Musumura et al. (1989) Plant Mol. Biol. 12: 123)); increaseddigestibility (e.g., modified storage proteins (U.S. application Ser.No. 10/053,410, filed Nov. 7, 2001); and thioredoxins (U.S. applicationSer. No. 10/005,429, filed Dec. 3, 2001)), the disclosures of which areherein incorporated by reference. The polynucleotides of the presentinvention can also be stacked with traits desirable for insect, diseaseor herbicide resistance (e.g., Bacillus thuringiensis toxic proteins(U.S. Pat. Nos. 5,366,892; 5,747,450; 5,737,514; 5723,756; 5,593,881;Geiser et al (1986) Gene 48:109); lectins (Van Damme et al. (1994) PlantMol. Biol. 24:825); fumonisin detoxification genes (U.S. Pat. No.5,792,931); avirulence and disease resistance genes (Jones et al. (1994)Science 266:789; Martin et al. (1993) Science 262:1432; Mindrinos et al.(1994) Cell 78:1089); acetolactate synthase (ALS) mutants that lead toherbicide resistance such as the S4 and/or Hra mutations; inhibitors ofglutamine synthase such as phosphinothricin or basta (e.g., bar gene);and glyphosate resistance (EPSPS gene)); and traits desirable forprocessing or process products such as high oil (e.g., U.S. Pat. No.6,232,529 ); modified oils (e.g., fatty acid desaturase genes (U.S. Pat.No. 5,952,544; WO 94/11516)); modified starches (e.g., ADPGpyrophosphorylases (AGPase), starch synthases (SS), starch branchingenzymes (SBE) and starch debranching enzymes (SDBE)); and polymers orbioplastics (e.g., U.S. Pat. No. 5,602,321; beta-ketothiolase,polyhydroxybutyrate synthase, and acetoacetyl-CoA reductase (Schubert etal. (1988) J. Bacteriol. 170:5837-5847) facilitate expression ofpolyhydroxyalkanoates (PHAs)), the disclosures of which are hereinincorporated by reference. One could also combine the polynucleotides ofthe present invention with polynucleotides providing agronomic traitssuch as male sterility (e.g., see U.S. Pat. No. 5,583,210), stalkstrength, flowering time, or transformation technology traits such ascell cycle regulation or gene targeting (e.g. WO 99/61619; WO 00/17364;WO 99/25821), the disclosures of which are herein incorporated byreference.

[0076] These stacked combinations can be created by any method includingbut not limited to cross breeding plants by any conventional or TopCrossmethodology, or genetic transformation. If the traits are stacked bygenetically transforming the plants, the polynucleotide sequences ofinterest can be combined at any time and in any order. For example, atransgenic plant comprising one or more desired traits can be used asthe target to introduce further traits by subsequent transformation. Thetraits can be introduced simultaneously in a co-transformation protocolwith the polynucleotides of interest provided by any combination oftransformation cassettes. For example, if two sequences will beintroduced, the two sequences can be contained in separatetransformation cassettes (trans) or contained on the same transformationcassette (cis). Expression of the sequences can be driven by the samepromoter or by different promoters. In certain cases, it may bedesirable to introduce a transformation cassette that will suppress theexpression of the polynucleotide of interest. This may be combine withany combination of other suppression cassettes or overexpressioncassettes to generate the desired combination of traits in the plant.

[0077] Furthermore, it is recognized that the methods of the inventionmay employ a nucleotide construct that is capable of directing, in atransformed plant, the expression of at least one protein, or at leastone RNA, such as, for example, an antisense RNA that is complementary toat least a portion of an mRNA of interest. Typically such a nucleotideconstruct is comprised of a coding sequence for a protein or an RNAoperably linked to 5′ and 3′ transcriptional regulatory regions.Alternatively, it is also recognized that the methods of the inventionmay employ a nucleotide construct that is not capable of directing, in atransformed plant, the expression of a protein or an RNA.

[0078] In addition, it is recognized that methods of the presentinvention do not depend on the incorporation of the entire nucleotideconstruct into the genome, only that the plant or cell thereof isaltered as a result of the introduction of the nucleotide construct intoa cell. In one embodiment of the invention, the genome may be alteredfollowing the introduction of the nucleotide construct into a cell. Forexample, the nucleotide construct, or any part thereof, may incorporateinto the genome of the plant. Alterations to the genome of a plant ofthe present invention include, but are not limited to, additions,deletions, and substitutions of nucleotides in the genome. While themethods of the present invention do not depend on additions, deletions,or substitutions of any particular number of nucleotides, it isrecognized that such additions, deletions, or substitutions comprise atleast one nucleotide.

[0079] The nucleotide constructs of the invention also encompassnucleotide constructs that may be employed in methods for altering ormutating a genomic nucleotide sequence in a plant, including, but notlimited to, chimeric vectors, chimeric mutational vectors, chimericrepair vectors, mixed-duplex oligonucleotides, self-complementarychimeric oligonucleotides, and recombinogenic oligonucleobases. Suchnucleotide constructs and methods of use, such as, for example,chimeraplasty, are known in the art. Chimeraplasty involves the use ofsuch nucleotide constructs to introduce site-specific changes into thesequence of genomic DNA within an organism. See, U.S. Pat. Nos.5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972; and 5,871,984;all of which are herein incorporated by reference. See also, WO98/49350, WO 99/07865, WO 99/25821, and Beetham et al. (1999) Proc.Natl. Acad. Sci. USA 96:8774-8778; herein incorporated by reference.

[0080] A number of promoters can be used in the practice of theinvention. The promoters can be selected based on the desired outcome.The nucleic acids can be combined with constitutive, tissue-preferred,or other promoters for expression in plants. Such constitutive promotersinclude, for example, the core promoter of the Rsyn7 promoter and otherconstitutive promoters disclosed in WO 99/43838 and U.S. Pat. No.6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2:163-171);ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 andChristensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last etal. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al. (1984)EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026), and thelike. Other constitutive promoters include, for example, U.S. Pat. Nos.5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680;5,268,463; and 5,608,142.

[0081] Chemical-regulated promoters can be used to modulate theexpression of a gene in a plant through the application of an exogenouschemical regulator. Depending upon the objective, the promoter can be achemical-inducible promoter, where application of the chemical inducesgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. For example, a chemicallyregulated promoter might be used to alter expression of the sequences ofthe invention prior to harvest. Application prior to harvest might allowthe benefits of the invention, including improved digestibility or gumextraction, without impinging normal plant growth or development.Chemical-inducible promoters are known in the art and include, but arenot limited to, the maize In2-2 promoter, which is activated bybenzenesulfonamide herbicide safeners, the maize GST promoter, which isactivated by hydrophobic electrophilic compounds that are used aspre-emergent herbicides, and the tobacco PR-1a promoter, which isactivated by salicylic acid. Other chemical-regulated promoters ofinterest include steroid-responsive promoters (see, for example, theglucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl.Acad. Sci. USA 88:10421-10425 and McNellis et al. (1998) Plant J.14(2):247-257) and tetracycline-inducible and tetracycline-repressiblepromoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet.227:229-237, and U.S. Pat. Nos. 5,814,618 and 5,789,156), hereinincorporated by reference.

[0082] Tissue-preferred promoters can be utilized to target modulationof polysaccharide synthase expression within a particular plant tissue.Tissue-preferred promoters include Yamamoto et al. (1997) Plant J.12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803;Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343; Russell et al.(1997) Transgenic Res. 6(2):157-168; Rinehart et al. (1996) PlantPhysiol. 112(3):1331-1341; Van Camp et al. (1996) Plant Physiol.112(2):525-535; Canevascini et al. (1996) Plant Physiol. 112(2):513-524;Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994)Results Probl. Cell Differ. 20:181-196; Orozco et al. (1993) Plant MolBiol. 23(6):1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J.4(3):495-505. Such promoters can be modified, if necessary, for weakexpression.

[0083] “Seed-preferred” promoters include both “seed-specific” promoters(those promoters active during seed development such as promoters ofseed storage proteins) as well as “seed-germinating” promoters (thosepromoters active during seed germination). See Thompson et al. (1989)BioEssays 10:108, herein incorporated by reference. Such seed-preferredpromoters include, but are not limited to, Cim1 (cytokinin-inducedmessage); cZ19B1 (maize 19 kDa zein); milps (myo-inositol-1-phosphatesynthase); and celA (cellulose synthase) (see the copending applicationentitled “Seed-Preferred Promoters,” U.S. application Ser. No.09/377,648, filed Aug. 19, 1999, herein incorporated by reference).Gama-zein is a preferred endosperm-specific promoter. Glob-1 is apreferred embryo-specific promoter. For dicots, seed-specific promotersinclude, but are not limited to, bean β-phaseolin, napin, β-conglycinin,soybean lectin, cruciferin, and the like. For monocots, seed-specificpromoters include, but are not limited to, maize 15 kDa zein, 22 kDazein, 27 kDa zein, g-zein, waxy, shrunken 1, shrunken 2, globulin 1,etc.

[0084] Root-preferred promoters are known and can be selected from themany available from the literature or isolated de novo from variouscompatible species. See, for example, Hire et al. (1992) Plant Mol.Biol. 20(2): 207-218 (soybean root-specific glutamine synthase gene);Keller and Baumgartner (1991) Plant Cell 3(10):1051-1061 (root-specificcontrol element in the GRP 1.8 gene of French bean); Sanger et al.(1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of themannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao etal. (1991) Plant Cell 3(1):11-22 (full-length cDNA clone encodingcytosolic glutamine synthase (GS), which is expressed in roots and rootnodules of soybean). See also Bogusz et al. (1990) Plant Cell2(7):633-641, where two root-specific promoters isolated from hemoglobingenes from the nitrogen-fixing nonlegume Parasponia andersonii and therelated non-nitrogen-fixing nonlegume Trema tomentosa are described. Thepromoters of these genes were linked to a β-glucuronidase reporter geneand introduced into both the nonlegume Nicotiana tabacum and the legumeLotus corniculatus, and in both instances root-specific promoteractivity was preserved. Leach and Aoyagi (1991) describe their analysisof the promoters of the highly expressed rolC and rolD root-inducinggenes of Agrobacterium rhizogenes (see Plant Science (Limerick)79(1):69-76). They concluded that enhancer and tissue-preferred DNAdeterminants are dissociated in those promoters. Teeri et al. (1989)used gene fusion to lacZ to show that the Agrobacterium T-DNA geneencoding octopine synthase is especially active in the epidermis of theroot tip and that the TR2′ gene is root specific in the intact plant andstimulated by wounding in leaf tissue, an especially desirablecombination of characteristics for use with an insecticidal orlarvicidal gene (see EMBO J. 8(2):343-350). The TR1′ gene, fused tonptII (neomycin phosphotransferase II) showed similar characteristics.Additional root-preferred promoters include the VfENOD-GRP3 genepromoter (Kuster et al. (1995) Plant Mol. Biol. 29(4):759-772); and rolBpromoter (Capana et al. (1994) Plant Mol. Biol. 25(4):681-691. See alsoU.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836;5,110,732; and 5,023,179; herein incorporated by reference.

[0085] Where low level expression is desired, weak promoters will beused. Generally, by “weak promoter” is intended a promoter that drivesexpression of a coding sequence at a low level. By low level is intendedat levels of about 1/1000 transcripts to about 1/100,000 transcripts toabout 1/500,000 transcripts. Alternatively, it is recognized that weakpromoters also encompass promoters that are expressed in only a fewcells and not in others to give a total low level of expression. Where apromoter is expressed at unacceptably high levels, portions of thepromoter sequence can be deleted or modified to decrease expressionlevels.

[0086] Such weak constitutive promoters include, for example, the corepromoter of the Rsyn7 promoter (WO 99/43838 and U.S. Pat. No.6,072,050), the core 35S CaMV promoter, and the like. Other constitutivepromoters include, for example, U.S. Pat. Nos. 5,608,149; 5,608,144;5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; and 5,608,142.See also, the copending application entitled “Constitutive MaizePromoters,” U.S. application Ser. No. 09/257,584, filed Feb. 25, 1999,and herein incorporated by reference.

[0087] Additional examples of promoters include the F3.7 promoter frommaize (Baszczynski, et al. (1997) Maydica 42:189-201); the soybeanalbumin promoter (U.S. Pat. No. 6,177,613), the beta conglycininpromoter (WO 91/13993), the Smas promoter, the cinnamyl alcoholdehydrogenase promoter (U.S. Pat. No. 5,683,439), the SCP1 promoter, theNos promoter, and the rubisco promoter. Yet more examples of promotersinclude the 1′- or 2′-promoter derived from T-DNA of Agrobacteriumtumefaciens, the histone H2B promoter (Nakayama et al. (1992) FEBS Lett30:167-170), the GRP1-8 promoter, and other transcription initiationregions from various plant genes known in the art.

[0088] Examples of promoters under developmental control includepromoters that initiate transcription preferentially in certain tissues,such as leaves, roots, fruits, seeds, or flowers. An exemplary promoteris the anther specific promoter 5126 (U.S. Pat. Nos. 5,689,049 and5,689,051). Examples of seed-preferred promoters include, but are notlimited to, 27 kD gamma zein promoter and waxy promoter, (Boronat et al.(1986) Plant Sci. 47:95-102, Reina et al. (1990) Nucleic Acids Res.18:6426, and Kloesgen et al. (1986) Mol Gen Genet 203:237-244, each ofwhich is herein incorporated by reference).

[0089] The methods of the invention involve introducing a nucleotideconstruct into a plant. By “introducing” is intended presenting to theplant the nucleotide construct in such a manner that the construct gainsaccess to the interior of a cell of the plant. The methods of theinvention do not depend on a particular method for introducing anucleotide construct to a plant, only that the nucleotide constructgains access to the interior of at least one cell of the plant. Methodsfor introducing nucleotide constructs into plants are known in the artincluding, but not limited to, stable transformation methods, transienttransformation methods, and virus-mediated methods.

[0090] By “stable transformation” is intended that the nucleotideconstruct introduced into a plant integrates into the genome of theplant and is capable of being inherited by progeny thereof. By“transient transformation” is intended that a nucleotide constructintroduced into a plant does not integrate into the genome of the plant.

[0091] Transformation protocols as well as protocols for introducingnucleotide sequences into plants may vary depending on the type of plantor plant cell, i.e., monocot or dicot, targeted for transformation.Suitable methods of introducing nucleotide sequences into plant cellsand subsequent insertion into the plant genome include microinjection(Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggset al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606,Agrobacterium-mediated transformation (Townsend et al., U.S. Pat. No.5,563,055; Zhao et al., U.S. Pat. No. 5,981,840), direct gene transfer(Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic particleacceleration (see, for example, Sanford et al., U.S. Pat. No. 4,945,050;Tomes et al., U.S. Pat. No. 5,879,918; Tomes et al., U.S. Pat. No.5,886,244; Bidney et al., U.S. Pat. No. 5,932,782; Tomes et al. (1995)“Direct DNA Transfer into Intact Plant Cells via MicroprojectileBombardment,” in Plant Cell, Tissue, and Organ Culture: FundamentalMethods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); and McCabeet al. (1988) Biotechnology 6:923-926). Also see Weissinger et al.(1988) Ann. Rev. Genet 22:421-477; Sanford et al. (1987) ParticulateScience and Technology 5:27-37 (onion); Christou et al. (1988) PlantPhysiol. 87:671-674 (soybean); McCabe et al. (1988) Bio/Technology6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol.27P:175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet.96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740(rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309(maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); Tomes,U.S. Pat. No. 5,240,855; Buising et al., U.S. Pat. Nos. 5,322,783 and5,324,646; Tomes et al. (1995) “Direct DNA Transfer into Intact PlantCells via Microprojectile Bombardment,” in Plant Cell, Tissue, and OrganCulture: Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin)(maize); Klein et al. (1988) Plant Physiol. 91:440-444 (maize); Fromm etal. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren etal. (1984) Nature (London) 311:763-764; Bowen et al., U.S. Pat. No.5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA84:5345-5349 (Liliaceae); De Wet et al. (1985) in The ExperimentalManipulation of Ovule Tissues, ed. Chapman et al. (Longman, N.Y.), pp.197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413(rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize viaAgrobacterium tumefaciens); all of which are herein incorporated byreference.

[0092] The nucleotide constructs of the invention may be introduced intoplants by contacting plants with a virus or viral nucleic acids.Generally, such methods involve incorporating a nucleotide construct ofthe invention within a viral DNA or RNA molecule. It is recognized thata polysaccharide synthase of the invention may be initially synthesizedas part of a viral polyprotein, which later may be processed byproteolysis in vivo or in vitro to produce the desired recombinantprotein. Further, it is recognized that promoters of the invention alsoencompass promoters utilized for transcription by viral RNA polymerases.Methods for introducing nucleotide constructs into plants and expressinga protein encoded therein, involving viral DNA or RNA molecules, areknown in the art. See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190,5,866,785, 5,589,367 and 5,316,931; herein incorporated by reference.

[0093] The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having the desired type of expression, forexample constitutive or tissue-preferred expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.

[0094] The nucleotide sequences encompassed by the present invention maybe used for transformation of any plant species, including, but notlimited to, monocots and dicots. Examples of plant species of interestinclude, but are not limited to, corn (Zea mays), Brassica sp. (e.g., B.napus, B. rapa, B. juncea), particularly those Brassica species usefulas sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa),rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet(e.g., pearl millet (Pennisetum glaucum), proso millet (Panicummiliaceum), foxtail millet (Setaria italica), finger millet (Eleusinecoracana)), sunflower (Helianthus annuus), safflower (Carthamustinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco(Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachishypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweetpotato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffeaspp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrustrees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis),banana (Musa spp.), avocado (Persea americana), fig (Ficus casica),guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (Anacardium occidentale),macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugarbeets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley,vegetables, ornamentals, and conifers.

[0095] Vegetables include tomatoes (Lycopersicon esculentum), lettuce(e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans(Phaseolus limensis), peas (Lathyrus spp.), and members of the genusCucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis),and muskmelon (C. melo). Ornamentals include azalea (Rhododendron spp.),hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis),roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.),petunias (Petunia hybrida), carnation (Dianthus caryophyllus),poinsettia (Euphorbia pulcherrima), and chrysanthemum.

[0096] Conifers that may be employed in practicing the present inventioninclude, for example, pines such as loblolly pine (Pinus taeda), slashpine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine(Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir(Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitkaspruce (Picea glauca); redwood (Sequoia sempervirens); true firs such assilver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedarssuch as Western red cedar (Thuja plicata) and Alaska yellow-cedar(Chamaecyparis nootkatensis). Preferably, plants of the presentinvention are crop plants (for example, corn, alfalfa, sunflower,Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet,tobacco, etc.), more preferably corn and soybean plants, yet morepreferably corn plants.

[0097] Plants of particular interest include grain plants that provideseeds of interest, oil-seed plants, and leguminous plants. Seeds ofinterest include grain seeds, such as corn, wheat, barley, rice,sorghum, rye, etc. Oil-seed plants include cotton, soybean, safflower,sunflower, Brassica, maize, alfalfa, palm, coconut, etc. Leguminousplants include beans and peas. Beans include guar, locust bean,fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, favabean, lentils, chickpea, etc.

[0098] This invention can be better understood by reference to thefollowing non-limited examples. It will be appreciated by those skilledin the art that other embodiments of the invention may be practicedwithout departing from the spirit and the scope of the invention asherein disclosed.

EXPERIMENTAL EXAMPLE 1 Particle Gun Transformation and Regeneration ofTransgenic Maize Plants

[0099] Immature maize embryos from greenhouse donor plants are bombardedwith a plasmid containing a polysaccharide synthase sequence of theinvention operably linked to a F3.7 promoter (Baszczynski, et al. (1997)Maydica 42:189-201) and the selectable marker gene PAT (Wohlleben et al.(1988) Gene 70:25-37), which confers resistance to the herbicideBialaphos. Alternatively, the selectable marker gene is provided on aseparate plasmid. Transformation is performed as follows. Media recipesfollow below.

Preparation of Target Tissue

[0100] The ears are husked and surface sterilized in 30% Clorox bleachplus 0.5% Micro detergent for 20 minutes, and rinsed two times withsterile water. The immature embryos are excised and placed embryo axisside down (scutellum side up), 25 embryos per plate, on 560Y medium for4 hours and then aligned within the 2.5-cm target zone in preparationfor bombardment.

Preparation of DNA

[0101] A plasmid vector comprising a nucleotide sequence of theinvention operably linked to a F3.7 promoter is made. This plasmid DNAplus plasmid DNA containing a PAT selectable marker is precipitated onto1.1 μm (average diameter) tungsten pellets using a CaCl₂ precipitationprocedure as follows:

[0102] 100 μl prepared tungsten particles in water

[0103] 10 μl (1 μg) DNA in Tris EDTA buffer (1 μg total DNA)

[0104] 100 μl 2.5 M CaC1₂

[0105] 10 μl 0.1 M spermidine

[0106] Each reagent is added sequentially to the tungsten particlesuspension, while maintained on the multitube vortexer. The finalmixture is sonicated briefly and allowed to incubate under constantvortexing for 10 minutes. After the precipitation period, the tubes arecentrifuged briefly, liquid removed, washed with 500 ml 100% ethanol,and centrifuged for 30 seconds. Again the liquid is removed, and 105 μl100% ethanol is added to the final tungsten particle pellet. Forparticle gun bombardment, the tungsten/DNA particles are brieflysonicated and 10 μl spotted onto the center of each macrocarrier andallowed to dry about 2 minutes before bombardment.

Particle Gun Treatment

[0107] The sample plates are bombarded at level #4 in particle gun#HE34-1 or #HE34-2. All samples receive a single shot at 650 PSI, with atotal of ten aliquots taken from each tube of prepared particles/DNA.

Subsequent Treatment

[0108] Following bombardment, the embryos are kept on 560Y medium for 2days, then transferred to 560R selection medium containing 3 mg/literBialaphos, and subcultured every 2 weeks. After approximately 10 weeksof selection, selection-resistant callus clones are transferred to 288Jmedium to initiate plant regeneration. Following somatic embryomaturation (2-4 weeks), well-developed somatic embryos are transferredto medium for germination and transferred to the lighted culture room.Approximately 7-10 days later, developing plantlets are transferred to272V hormone-free medium in tubes for 7-10 days until plantlets are wellestablished. Plants are then transferred to inserts in flats (equivalentto 2.5″ pot) containing potting soil and grown for 1 week in a growthchamber, subsequently grown an additional 1-2 weeks in the greenhouse,then transferred to classic 600 pots (1.6 gallon) and grown to maturity.Plants are monitored and scored for altered polysaccharide synthaseactivity.

Bombardment and Culture Media

[0109] Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts (SIGMAC-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000×SIGMA-1511), 0.5 mg/lthiamine HCl, 120.0 g/l sucrose, 1.0 mg/l 2,4-D, and 2.88 g/l L-proline(brought to volume with D-I H₂0 following adjustment to pH 5.8 withKOH); 2.0 g/l Gelrite (added after bringing to volume with D-I H₂0); and8.5 mg/l silver nitrate (added after sterilizing the medium and coolingto room temperature). Selection medium (560R) comprises 4.0 g/l N6 basalsalts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000×SIGMA-1511),0.5 mg/l thiamine HCl, 30.0 g/l sucrose, and 2.0 mg/l 2,4-D (brought tovolume with D-I H₂0 following adjustment to pH 5.8 with KOH); 3.0 g/lGelrite (added after bringing to volume with D-I H₂0); and 0.85 mg/lsilver nitrate and 3.0 mg/l bialaphos (both added after sterilizing themedium and cooling to room temperature).

[0110] Plant regeneration medium (288J) comprises 4.3 g/l MS salts(GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100 gnicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40g/l glycine brought to volume with polished D-I H₂0) (Murashige andSkoog (1962) Physiol. Plant 15:473), 100 mg/l myo-inositol, 0.5 mg/lzeatin, 60 g/l sucrose, and 1.0 ml/l of 0.1 mM abscisic acid (brought tovolume with polished D-I H₂0 after adjusting to pH 5.6); 3.0 g/l Gelrite(added after bringing to volume with D-I H₂0); and 1.0 mg/l indoleaceticacid and 3.0 mg/l bialaphos (added after sterilizing the medium andcooling to 60° C.). Hormone-free medium (272V) comprises 4.3 g/l MSsalts (GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g/lnicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40g/l glycine brought to volume with polished D-I H₂0), 0.1 g/lmyo-inositol, and 40.0 g/l sucrose (brought to volume with polished D-IH₂0 after adjusting pH to 5.6); and 6 g/l bacto-agar (added afterbringing to volume with polished D-I H₂0), sterilized and cooled to 60°C.

EXAMPLE 2 Agrobacterium-Mediated Transformation of Maize

[0111] For Agrobacterium-mediated transformation of maize withpolysaccharide synthase gene(s) or nucleotide sequence(s) of theinvention, preferably the method of Zhao is employed (U.S. Pat. No.5,981,840, and PCT patent publication WO98/32326; the contents of whichare hereby incorporated by reference). Briefly, immature embryos areisolated from maize and the embryos contacted with a suspension ofAgrobacterium, where the bacteria are capable of transferring thepolysaccharide synthase gene(s) or nucleotide sequence(s) to at leastone cell of at least one of the immature embryos (step 1: the infectionstep). In this step the immature embryos are preferably immersed in anAgrobacterium suspension for the initiation of inoculation. The embryosare co-cultured for a time with the Agrobacterium (step 2: theco-cultivation step). Preferably the immature embryos are cultured onsolid medium following the infection step. Following this co-cultivationperiod an optional “resting” step is contemplated. In this resting step,the embryos are incubated in the presence of at least one antibioticknown to inhibit the growth of Agrobacterium without the addition of aselective agent for plant transformants (step 3: resting step).Preferably the immature embryos are cultured on solid medium withantibiotic, but without a selecting agent, for elimination ofAgrobacterium and for a resting phase for the infected cells. Next,inoculated embryos are cultured on medium containing a selective agentand growing transformed callus is recovered (step 4: the selectionstep). Preferably, the immature embryos are cultured on solid mediumwith a selective agent resulting in the selective growth of transformedcells. The callus is then regenerated into plants (step 5: theregeneration step), and preferably calli grown on selective medium arecultured on solid medium to regenerate the plants. Regeneratedtransgenic plants are then monitored for altered polysaccharide synthaseactivity.

EXAMPLE 3 Soybean Embryo Transformation Example

[0112] Soybean embryos are bombarded with a plasmid containing apolysaccharide synthase gene or nucleotide sequence of the inventionoperably linked to a soybean albumin promoter (U.S. Pat. No. 6,177,613)as follows. To induce somatic embryos, cotyledons, 3-5 mm in lengthdissected from surface-sterilized, immature seeds of the soybeancultivar A2872, are cultured in the light or dark at 26° C. on anappropriate agar medium for six to ten weeks. Somatic embryos producingsecondary embryos are then excised and placed into a suitable liquidmedium. After repeated selection for clusters of somatic embryos thatmultiplied as early, globular-staged embryos, the suspensions aremaintained as described below.

[0113] Soybean embryogenic suspension cultures can maintained in 35 mlliquid media on a rotary shaker, 150 rpm, at 26° C. with florescentlights on a 16:8 hour day/night schedule. Cultures are subcultured everytwo weeks by inoculating approximately 35 mg of tissue into 35 ml ofliquid medium.

[0114] Soybean embryogenic suspension cultures may then be transformedby the method of particle gun bombardment (Klein et al. (1987)Nature(London) 327:70-73, U.S. Pat. No. 4,945,050). A Du Pont BiolisticPDS1000/HE instrument (helium retrofit) can be used for thesetransformations.

[0115] A selectable marker gene that can be used to facilitate soybeantransformation is a transgene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al. (1983) Gene 25:179-188), and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The expression cassette comprising a polysaccharidesynthase nucleotide sequence operably linked to the soybean albuminpromoter can be isolated as a restriction fragment. This fragment canthen be inserted into a unique restriction site of the vector carryingthe marker gene.

[0116] To 50 μl of a 60 mg/ml 1 μm gold particle suspension is added (inorder): 5 μl DNA (1 μg/μl), 20 μl spermidine (0.1 M), and 50 μl CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μl 70% ethanol andresuspended in 40 μl of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five microliters ofthe DNA-coated gold particles are then loaded on each macro carrierdisk.

[0117] Approximately 300-400 mg of a two-week-old suspension culture isplaced in an empty 60×15 mm petri dish and the residual liquid removedfrom the tissue with a pipette. For each transformation experiment,approximately 5-10 plates of tissue are normally bombarded. Membranerupture pressure is set at 1100 psi, and the chamber is evacuated to avacuum of 28 inches mercury. The tissue is placed approximately 3.5inches away from the retaining screen and bombarded three times.Following bombardment, the tissue can be divided in half and placed backinto liquid and cultured as described above.

[0118] Five to seven days post bombardment, the liquid media may beexchanged with fresh media, and eleven to twelve days post-bombardmentwith fresh media containing 50 mg/ml hygromycin. This selective mediacan be refreshed weekly. Seven to eight weeks post-bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos. Regenerated transgenic plants are thenmonitored for altered polysaccharide synthase activity.

[0119] All publications and patent applications mentioned in thespecification are indicative of the level of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

[0120] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended embodiments.

1 30 1 1910 DNA Zea mays 1 tcgacccacg cgtccggtcc gttcatctgc tgtcatgtcaaatggcccct tcatcctcaa 60 cctcgactgt gatcactatg tctacaactc gcaagctttccgcgaaggga tgtgcttcat 120 gatggaccgt ggtggcgacc gcattggtta tgtccagttcccgcagcggt ttgagggcat 180 cgatccatca gatcgctatg ccaaccacaa caccgtcttcttcgacgtca acatgcgcgc 240 gctggatggt ctcatgggac cagtctatgt tggcactggctgccttttcc gccgtgttgc 300 cctatatgga tttgaccctc cgcgctccaa ggagcacggtggctgctgca gctgttgctt 360 cccccagaga cgcaagatca aagcttcagc cgctgcaccggaggagaccc gggctctaag 420 gatggcagac ttcgacgagg atgaaatgaa catgtcgtcgttccccaaga agtttggtaa 480 ctcgagcttc ctcatcgact ccattccgat tgctgagttccaagggcgcc cgcttgctga 540 tcaccctggt gtcaagaacg gccgccctcc cggtgctctcactgtccccc gtgaccttct 600 ggatgcatcc acagtcgctg aggccgtcag tgtcatctcatgctggtacg aagacaagac 660 cgagtggggc caccgtgttg gttggatcta tggctcggtgacggaggatg tggtcactgg 720 gtaccggatg cacaaccggg gttggaagtc ggtgtactgtgtcaccaagc gtgacgcctt 780 ccacggcacc gcgcccatca acctgactga ccgtctccaccaggtgctcc ggtgggctac 840 tggatcagtg gagatcttct tctcccgcaa caacgcgctgctggcgagcc gcagaatgaa 900 gttcttgcag aggatcgcgt acctgaacgt gggtatctacccgttcacgt ccatcttcct 960 gatcgtctac tgcttcctgc cggcgctgtc gctgttctcggggcagttca tcgtgaagac 1020 gctgaacgtg acgttcctga cgtacctgct ggtgatcacgctgacgctgt gcctgctggc 1080 ggtgctggag atcaagtggt cggggatcag tctggaggagtggtggcgga acgagcagtt 1140 ctggctgatc ggcggcacga gcgcgcacct ggcggccgtgctgcagggcc tgctgaaggt 1200 ggtggcgggc atcgagatct ccttcacgct gacgtccaagtcgggcggcg acgacgtgga 1260 cgacgagttc gcggacctgt acatcgtcaa gtggacgtcgctgatgatcc cgcccatcgt 1320 gatcatgatg gtgaacctga tcggcatcgc ggtcgggttcagccgcacca tctacagcga 1380 gatcccgcag tggagcaagc tgctgggcgg cgtcttcttcagcttctggg tgctggcgca 1440 cctgtacccg ttcgccaagg gcctgatggg gcggaggggccgcacgccaa ccatcgtctt 1500 cgtctgggcg ggcctcctct ccatcaccat ctcgctgctgtgggtggcca tcaacccgcc 1560 gtcccagaac cagcagattg gtgggtcgtt cacattcccgtgaaagctct ctgggccaat 1620 ggcggattca tgcatgcttc gtcgcagtgg gatccttgcgttgctctgca tcagttcctg 1680 gttgcagcgg ttcaatatct gaggacgaag ctgctgggggaatgtgggat ccctgacttg 1740 tcgaaactgg cttacttttt ttgttgtcgg aagcttgataaatactctta tgatgagcta 1800 tagtagtagt tcttttgttc ttttgttttt gctatatataaaatacatgt tctggtccct 1860 taaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa 1910 2 522 PRT Zea mays 2 Met Ser Asn Gly Pro Phe Ile Leu AsnLeu Asp Cys Asp His Tyr Val 1 5 10 15 Tyr Asn Ser Gln Ala Phe Arg GluGly Met Cys Phe Met Met Asp Arg 20 25 30 Gly Gly Asp Arg Ile Gly Tyr ValGln Phe Pro Gln Arg Phe Glu Gly 35 40 45 Ile Asp Pro Ser Asp Arg Tyr AlaAsn His Asn Thr Val Phe Phe Asp 50 55 60 Val Asn Met Arg Ala Leu Asp GlyLeu Met Gly Pro Val Tyr Val Gly 65 70 75 80 Thr Gly Cys Leu Phe Arg ArgVal Ala Leu Tyr Gly Phe Asp Pro Pro 85 90 95 Arg Ser Lys Glu His Gly GlyCys Cys Ser Cys Cys Phe Pro Gln Arg 100 105 110 Arg Lys Ile Lys Ala SerAla Ala Ala Pro Glu Glu Thr Arg Ala Leu 115 120 125 Arg Met Ala Asp PheAsp Glu Asp Glu Met Asn Met Ser Ser Phe Pro 130 135 140 Lys Lys Phe GlyAsn Ser Ser Phe Leu Ile Asp Ser Ile Pro Ile Ala 145 150 155 160 Glu PheGln Gly Arg Pro Leu Ala Asp His Pro Gly Val Lys Asn Gly 165 170 175 ArgPro Pro Gly Ala Leu Thr Val Pro Arg Asp Leu Leu Asp Ala Ser 180 185 190Thr Val Ala Glu Ala Val Ser Val Ile Ser Cys Trp Tyr Glu Asp Lys 195 200205 Thr Glu Trp Gly His Arg Val Gly Trp Ile Tyr Gly Ser Val Thr Glu 210215 220 Asp Val Val Thr Gly Tyr Arg Met His Asn Arg Gly Trp Lys Ser Val225 230 235 240 Tyr Cys Val Thr Lys Arg Asp Ala Phe His Gly Thr Ala ProIle Asn 245 250 255 Leu Thr Asp Arg Leu His Gln Val Leu Arg Trp Ala ThrGly Ser Val 260 265 270 Glu Ile Phe Phe Ser Arg Asn Asn Ala Leu Leu AlaSer Arg Arg Met 275 280 285 Lys Phe Leu Gln Arg Ile Ala Tyr Leu Asn ValGly Ile Tyr Pro Phe 290 295 300 Thr Ser Ile Phe Leu Ile Val Tyr Cys PheLeu Pro Ala Leu Ser Leu 305 310 315 320 Phe Ser Gly Gln Phe Ile Val LysThr Leu Asn Val Thr Phe Leu Thr 325 330 335 Tyr Leu Leu Val Ile Thr LeuThr Leu Cys Leu Leu Ala Val Leu Glu 340 345 350 Ile Lys Trp Ser Gly IleSer Leu Glu Glu Trp Trp Arg Asn Glu Gln 355 360 365 Phe Trp Leu Ile GlyGly Thr Ser Ala His Leu Ala Ala Val Leu Gln 370 375 380 Gly Leu Leu LysVal Val Ala Gly Ile Glu Ile Ser Phe Thr Leu Thr 385 390 395 400 Ser LysSer Gly Gly Asp Asp Val Asp Asp Glu Phe Ala Asp Leu Tyr 405 410 415 IleVal Lys Trp Thr Ser Leu Met Ile Pro Pro Ile Val Ile Met Met 420 425 430Val Asn Leu Ile Gly Ile Ala Val Gly Phe Ser Arg Thr Ile Tyr Ser 435 440445 Glu Ile Pro Gln Trp Ser Lys Leu Leu Gly Gly Val Phe Phe Ser Phe 450455 460 Trp Val Leu Ala His Leu Tyr Pro Phe Ala Lys Gly Leu Met Gly Arg465 470 475 480 Arg Gly Arg Thr Pro Thr Ile Val Phe Val Trp Ala Gly LeuLeu Ser 485 490 495 Ile Thr Ile Ser Leu Leu Trp Val Ala Ile Asn Pro ProSer Gln Asn 500 505 510 Gln Gln Ile Gly Gly Ser Phe Thr Phe Pro 515 5203 1569 DNA Zea mays 3 atgtcaaatg gccccttcat cctcaacctc gactgtgatcactatgtcta caactcgcaa 60 gctttccgcg aagggatgtg cttcatgatg gaccgtggtggcgaccgcat tggttatgtc 120 cagttcccgc agcggtttga gggcatcgat ccatcagatcgctatgccaa ccacaacacc 180 gtcttcttcg acgtcaacat gcgcgcgctg gatggtctcatgggaccagt ctatgttggc 240 actggctgcc ttttccgccg tgttgcccta tatggatttgaccctccgcg ctccaaggag 300 cacggtggct gctgcagctg ttgcttcccc cagagacgcaagatcaaagc ttcagccgct 360 gcaccggagg agacccgggc tctaaggatg gcagacttcgacgaggatga aatgaacatg 420 tcgtcgttcc ccaagaagtt tggtaactcg agcttcctcatcgactccat tccgattgct 480 gagttccaag ggcgcccgct tgctgatcac cctggtgtcaagaacggccg ccctcccggt 540 gctctcactg tcccccgtga ccttctggat gcatccacagtcgctgaggc cgtcagtgtc 600 atctcatgct ggtacgaaga caagaccgag tggggccaccgtgttggttg gatctatggc 660 tcggtgacgg aggatgtggt cactgggtac cggatgcacaaccggggttg gaagtcggtg 720 tactgtgtca ccaagcgtga cgccttccac ggcaccgcgcccatcaacct gactgaccgt 780 ctccaccagg tgctccggtg ggctactgga tcagtggagatcttcttctc ccgcaacaac 840 gcgctgctgg cgagccgcag aatgaagttc ttgcagaggatcgcgtacct gaacgtgggt 900 atctacccgt tcacgtccat cttcctgatc gtctactgcttcctgccggc gctgtcgctg 960 ttctcggggc agttcatcgt gaagacgctg aacgtgacgttcctgacgta cctgctggtg 1020 atcacgctga cgctgtgcct gctggcggtg ctggagatcaagtggtcggg gatcagtctg 1080 gaggagtggt ggcggaacga gcagttctgg ctgatcggcggcacgagcgc gcacctggcg 1140 gccgtgctgc agggcctgct gaaggtggtg gcgggcatcgagatctcctt cacgctgacg 1200 tccaagtcgg gcggcgacga cgtggacgac gagttcgcggacctgtacat cgtcaagtgg 1260 acgtcgctga tgatcccgcc catcgtgatc atgatggtgaacctgatcgg catcgcggtc 1320 gggttcagcc gcaccatcta cagcgagatc ccgcagtggagcaagctgct gggcggcgtc 1380 ttcttcagct tctgggtgct ggcgcacctg tacccgttcgccaagggcct gatggggcgg 1440 aggggccgca cgccaaccat cgtcttcgtc tgggcgggcctcctctccat caccatctcg 1500 ctgctgtggg tggccatcaa cccgccgtcc cagaaccagcagattggtgg gtcgttcaca 1560 ttcccgtga 1569 4 522 PRT Zea mays 4 Met SerAsn Gly Pro Phe Ile Leu Asn Leu Asp Cys Asp His Tyr Val 1 5 10 15 TyrAsn Ser Gln Ala Phe Arg Glu Gly Met Cys Phe Met Met Asp Arg 20 25 30 GlyGly Asp Arg Ile Gly Tyr Val Gln Phe Pro Gln Arg Phe Glu Gly 35 40 45 IleAsp Pro Ser Asp Arg Tyr Ala Asn His Asn Thr Val Phe Phe Asp 50 55 60 ValAsn Met Arg Ala Leu Asp Gly Leu Met Gly Pro Val Tyr Val Gly 65 70 75 80Thr Gly Cys Leu Phe Arg Arg Val Ala Leu Tyr Gly Phe Asp Pro Pro 85 90 95Arg Ser Lys Glu His Gly Gly Cys Cys Ser Cys Cys Phe Pro Gln Arg 100 105110 Arg Lys Ile Lys Ala Ser Ala Ala Ala Pro Glu Glu Thr Arg Ala Leu 115120 125 Arg Met Ala Asp Phe Asp Glu Asp Glu Met Asn Met Ser Ser Phe Pro130 135 140 Lys Lys Phe Gly Asn Ser Ser Phe Leu Ile Asp Ser Ile Pro IleAla 145 150 155 160 Glu Phe Gln Gly Arg Pro Leu Ala Asp His Pro Gly ValLys Asn Gly 165 170 175 Arg Pro Pro Gly Ala Leu Thr Val Pro Arg Asp LeuLeu Asp Ala Ser 180 185 190 Thr Val Ala Glu Ala Val Ser Val Ile Ser CysTrp Tyr Glu Asp Lys 195 200 205 Thr Glu Trp Gly His Arg Val Gly Trp IleTyr Gly Ser Val Thr Glu 210 215 220 Asp Val Val Thr Gly Tyr Arg Met HisAsn Arg Gly Trp Lys Ser Val 225 230 235 240 Tyr Cys Val Thr Lys Arg AspAla Phe His Gly Thr Ala Pro Ile Asn 245 250 255 Leu Thr Asp Arg Leu HisGln Val Leu Arg Trp Ala Thr Gly Ser Val 260 265 270 Glu Ile Phe Phe SerArg Asn Asn Ala Leu Leu Ala Ser Arg Arg Met 275 280 285 Lys Phe Leu GlnArg Ile Ala Tyr Leu Asn Val Gly Ile Tyr Pro Phe 290 295 300 Thr Ser IlePhe Leu Ile Val Tyr Cys Phe Leu Pro Ala Leu Ser Leu 305 310 315 320 PheSer Gly Gln Phe Ile Val Lys Thr Leu Asn Val Thr Phe Leu Thr 325 330 335Tyr Leu Leu Val Ile Thr Leu Thr Leu Cys Leu Leu Ala Val Leu Glu 340 345350 Ile Lys Trp Ser Gly Ile Ser Leu Glu Glu Trp Trp Arg Asn Glu Gln 355360 365 Phe Trp Leu Ile Gly Gly Thr Ser Ala His Leu Ala Ala Val Leu Gln370 375 380 Gly Leu Leu Lys Val Val Ala Gly Ile Glu Ile Ser Phe Thr LeuThr 385 390 395 400 Ser Lys Ser Gly Gly Asp Asp Val Asp Asp Glu Phe AlaAsp Leu Tyr 405 410 415 Ile Val Lys Trp Thr Ser Leu Met Ile Pro Pro IleVal Ile Met Met 420 425 430 Val Asn Leu Ile Gly Ile Ala Val Gly Phe SerArg Thr Ile Tyr Ser 435 440 445 Glu Ile Pro Gln Trp Ser Lys Leu Leu GlyGly Val Phe Phe Ser Phe 450 455 460 Trp Val Leu Ala His Leu Tyr Pro PheAla Lys Gly Leu Met Gly Arg 465 470 475 480 Arg Gly Arg Thr Pro Thr IleVal Phe Val Trp Ala Gly Leu Leu Ser 485 490 495 Ile Thr Ile Ser Leu LeuTrp Val Ala Ile Asn Pro Pro Ser Gln Asn 500 505 510 Gln Gln Ile Gly GlySer Phe Thr Phe Pro 515 520 5 1673 DNA Zea mays 5 tcttcgccgc cgcctacgcggcctggatgc gcgcccgcct cgactacctc gcgccgccgc 60 tgcagttcct aaccaacgcctgcgtcctcc tcttcctggt ccagagcgtc gaccgcctcg 120 tgctctgcct cggctgcttctggatcaagc tcaagggcgt caggcccgtg ccgccgctgc 180 ccgccgacaa ggaggacgtcgaggccggtc ccgacggcgt ccccatggtg ctcgtgcaga 240 tgcccatgtg caatgagagagaggtgtatc aacaatcaat tgcggccgtg tgcaaccttg 300 actggcccaa atccaacttcttggtccaag tgttggatga ctccgacgac ccactcacaa 360 aggctctaat cagagaagaagtggccaaat ggcaacagca gggtgcccgg attgtgtacc 420 ggcaccgggt gatccgggatggctacaagg ctggaaacct gaaatcagcc atgaactgca 480 gttacgtgaa agactatgagttcgttgtca tcttcgatgc tgatttccaa ccacaggcgg 540 acttcctgaa gcgcaccgtgccccatttca agggaaagga tgacgtcggg ttggttcagg 600 cgagatggtc gttcgtaaacaaggatgaga acttgctgac caggcttcag aacataaatc 660 tttgcttcca cttcgaggtggagcagcagg tgaacggggc gtttctcaac ttcttcgggt 720 tcaatggcac cgcgggagtctggagaatca aggcgcttga ggagtctgga ggatggatgg 780 agaggacgac ggtggaggacatggacatag ctgttcgagc gcacctcaaa gggtggaagt 840 ttctctttct aaacgatgtcgagtgtcagt gtgaattgcc agaatcgtat gaagcgtaca 900 gaaagcagca gcaccggtggcactcaggtc ccatgcaatt gtttaggctc tgctttgtgg 960 atataatcaa atctaagatcggtttctgga agaagttcaa cctcatattc ctcttcttcc 1020 tgctccggaa gctgatactacccttctact ccttcaccct cttctgcatc atcctcccga 1080 tgacgatgtt cgtgccggaggccgagctcc ccgactgggt ggtgtgctac gtcccggccc 1140 tgatgtccct gctgaacatcctgccgtccc ccaagtcgtt ccccttcatc atcccgtacc 1200 tgctcttcga gaacaccatgtccgtgacca agttcaacgc gatgatctcc gggctgttcc 1260 agctggggag cgcgtacgagtgggtggtga ccaagaagtc gggccgctcg tcggagggcg 1320 acctcatcgc gctggccccgcccaaggagc ctgtgaagca cgcgacgagg acgggctccg 1380 cgccgaacct cgacgccgtcgccaaggagg agcaacagca gcagcagctg gcggcgtcga 1440 ggaaggacgc cgccgcgaagaagaaggaga agcacaaccg gatatacaag aaggagctgg 1500 cgctgtcgat gctgctcctgaccgcggccg cccggagcct gctgtcgaag catggcatac 1560 acttctactt cctcctgttccagggcgtgt ccttcttgct agtaggcctt gacctcatag 1620 gcgagcaagt cgagtgaaatgtgtataaca ggaaactgat acgtgtggga aaa 1673 6 536 PRT Zea mays 6 Met ArgAla Arg Leu Asp Tyr Leu Ala Pro Pro Leu Gln Phe Leu Thr 1 5 10 15 AsnAla Cys Val Leu Leu Phe Leu Val Gln Ser Val Asp Arg Leu Val 20 25 30 LeuCys Leu Gly Cys Phe Trp Ile Lys Leu Lys Gly Val Arg Pro Val 35 40 45 ProPro Leu Pro Ala Asp Lys Glu Asp Val Glu Ala Gly Pro Asp Gly 50 55 60 ValPro Met Val Leu Val Gln Met Pro Met Cys Asn Glu Arg Glu Val 65 70 75 80Tyr Gln Gln Ser Ile Ala Ala Val Cys Asn Leu Asp Trp Pro Lys Ser 85 90 95Asn Phe Leu Val Gln Val Leu Asp Asp Ser Asp Asp Pro Leu Thr Lys 100 105110 Ala Leu Ile Arg Glu Glu Val Ala Lys Trp Gln Gln Gln Gly Ala Arg 115120 125 Ile Val Tyr Arg His Arg Val Ile Arg Asp Gly Tyr Lys Ala Gly Asn130 135 140 Leu Lys Ser Ala Met Asn Cys Ser Tyr Val Lys Asp Tyr Glu PheVal 145 150 155 160 Val Ile Phe Asp Ala Asp Phe Gln Pro Gln Ala Asp PheLeu Lys Arg 165 170 175 Thr Val Pro His Phe Lys Gly Lys Asp Asp Val GlyLeu Val Gln Ala 180 185 190 Arg Trp Ser Phe Val Asn Lys Asp Glu Asn LeuLeu Thr Arg Leu Gln 195 200 205 Asn Ile Asn Leu Cys Phe His Phe Glu ValGlu Gln Gln Val Asn Gly 210 215 220 Ala Phe Leu Asn Phe Phe Gly Phe AsnGly Thr Ala Gly Val Trp Arg 225 230 235 240 Ile Lys Ala Leu Glu Glu SerGly Gly Trp Met Glu Arg Thr Thr Val 245 250 255 Glu Asp Met Asp Ile AlaVal Arg Ala His Leu Lys Gly Trp Lys Phe 260 265 270 Leu Phe Leu Asn AspVal Glu Cys Gln Cys Glu Leu Pro Glu Ser Tyr 275 280 285 Glu Ala Tyr ArgLys Gln Gln His Arg Trp His Ser Gly Pro Met Gln 290 295 300 Leu Phe ArgLeu Cys Phe Val Asp Ile Ile Lys Ser Lys Ile Gly Phe 305 310 315 320 TrpLys Lys Phe Asn Leu Ile Phe Leu Phe Phe Leu Leu Arg Lys Leu 325 330 335Ile Leu Pro Phe Tyr Ser Phe Thr Leu Phe Cys Ile Ile Leu Pro Met 340 345350 Thr Met Phe Val Pro Glu Ala Glu Leu Pro Asp Trp Val Val Cys Tyr 355360 365 Val Pro Ala Leu Met Ser Leu Leu Asn Ile Leu Pro Ser Pro Lys Ser370 375 380 Phe Pro Phe Ile Ile Pro Tyr Leu Leu Phe Glu Asn Thr Met SerVal 385 390 395 400 Thr Lys Phe Asn Ala Met Ile Ser Gly Leu Phe Gln LeuGly Ser Ala 405 410 415 Tyr Glu Trp Val Val Thr Lys Lys Ser Gly Arg SerSer Glu Gly Asp 420 425 430 Leu Ile Ala Leu Ala Pro Pro Lys Glu Pro ValLys His Ala Thr Arg 435 440 445 Thr Gly Ser Ala Pro Asn Leu Asp Ala ValAla Lys Glu Glu Gln Gln 450 455 460 Gln Gln Gln Leu Ala Ala Ser Arg LysAsp Ala Ala Ala Lys Lys Lys 465 470 475 480 Glu Lys His Asn Arg Ile TyrLys Lys Glu Leu Ala Leu Ser Met Leu 485 490 495 Leu Leu Thr Ala Ala AlaArg Ser Leu Leu Ser Lys His Gly Ile His 500 505 510 Phe Tyr Phe Leu LeuPhe Gln Gly Val Ser Phe Leu Leu Val Gly Leu 515 520 525 Asp Leu Ile GlyGlu Gln Val Glu 530 535 7 1611 DNA Zea mays 7 atgcgcgccc gcctcgactacctcgcgccg ccgctgcagt tcctaaccaa cgcctgcgtc 60 ctcctcttcc tggtccagagcgtcgaccgc ctcgtgctct gcctcggctg cttctggatc 120 aagctcaagg gcgtcaggcccgtgccgccg ctgcccgccg acaaggagga cgtcgaggcc 180 ggtcccgacg gcgtccccatggtgctcgtg cagatgccca tgtgcaatga gagagaggtg 240 tatcaacaat caattgcggccgtgtgcaac cttgactggc ccaaatccaa cttcttggtc 300 caagtgttgg atgactccgacgacccactc acaaaggctc taatcagaga agaagtggcc 360 aaatggcaac agcagggtgcccggattgtg taccggcacc gggtgatccg ggatggctac 420 aaggctggaa acctgaaatcagccatgaac tgcagttacg tgaaagacta tgagttcgtt 480 gtcatcttcg atgctgatttccaaccacag gcggacttcc tgaagcgcac cgtgccccat 540 ttcaagggaa aggatgacgtcgggttggtt caggcgagat ggtcgttcgt aaacaaggat 600 gagaacttgc tgaccaggcttcagaacata aatctttgct tccacttcga ggtggagcag 660 caggtgaacg gggcgtttctcaacttcttc gggttcaatg gcaccgcggg agtctggaga 720 atcaaggcgc ttgaggagtctggaggatgg atggagagga cgacggtgga ggacatggac 780 atagctgttc gagcgcacctcaaagggtgg aagtttctct ttctaaacga tgtcgagtgt 840 cagtgtgaat tgccagaatcgtatgaagcg tacagaaagc agcagcaccg gtggcactca 900 ggtcccatgc aattgtttaggctctgcttt gtggatataa tcaaatctaa gatcggtttc 960 tggaagaagt tcaacctcatattcctcttc ttcctgctcc ggaagctgat actacccttc 1020 tactccttca ccctcttctgcatcatcctc ccgatgacga tgttcgtgcc ggaggccgag 1080 ctccccgact gggtggtgtgctacgtcccg gccctgatgt ccctgctgaa catcctgccg 1140 tcccccaagt cgttccccttcatcatcccg tacctgctct tcgagaacac catgtccgtg 1200 accaagttca acgcgatgatctccgggctg ttccagctgg ggagcgcgta cgagtgggtg 1260 gtgaccaaga agtcgggccgctcgtcggag ggcgacctca tcgcgctggc cccgcccaag 1320 gagcctgtga agcacgcgacgaggacgggc tccgcgccga acctcgacgc cgtcgccaag 1380 gaggagcaac agcagcagcagctggcggcg tcgaggaagg acgccgccgc gaagaagaag 1440 gagaagcaca accggatatacaagaaggag ctggcgctgt cgatgctgct cctgaccgcg 1500 gccgcccgga gcctgctgtcgaagcatggc atacacttct acttcctcct gttccagggc 1560 gtgtccttct tgctagtaggccttgacctc ataggcgagc aagtcgagtg a 1611 8 536 PRT Zea mays 8 Met Arg AlaArg Leu Asp Tyr Leu Ala Pro Pro Leu Gln Phe Leu Thr 1 5 10 15 Asn AlaCys Val Leu Leu Phe Leu Val Gln Ser Val Asp Arg Leu Val 20 25 30 Leu CysLeu Gly Cys Phe Trp Ile Lys Leu Lys Gly Val Arg Pro Val 35 40 45 Pro ProLeu Pro Ala Asp Lys Glu Asp Val Glu Ala Gly Pro Asp Gly 50 55 60 Val ProMet Val Leu Val Gln Met Pro Met Cys Asn Glu Arg Glu Val 65 70 75 80 TyrGln Gln Ser Ile Ala Ala Val Cys Asn Leu Asp Trp Pro Lys Ser 85 90 95 AsnPhe Leu Val Gln Val Leu Asp Asp Ser Asp Asp Pro Leu Thr Lys 100 105 110Ala Leu Ile Arg Glu Glu Val Ala Lys Trp Gln Gln Gln Gly Ala Arg 115 120125 Ile Val Tyr Arg His Arg Val Ile Arg Asp Gly Tyr Lys Ala Gly Asn 130135 140 Leu Lys Ser Ala Met Asn Cys Ser Tyr Val Lys Asp Tyr Glu Phe Val145 150 155 160 Val Ile Phe Asp Ala Asp Phe Gln Pro Gln Ala Asp Phe LeuLys Arg 165 170 175 Thr Val Pro His Phe Lys Gly Lys Asp Asp Val Gly LeuVal Gln Ala 180 185 190 Arg Trp Ser Phe Val Asn Lys Asp Glu Asn Leu LeuThr Arg Leu Gln 195 200 205 Asn Ile Asn Leu Cys Phe His Phe Glu Val GluGln Gln Val Asn Gly 210 215 220 Ala Phe Leu Asn Phe Phe Gly Phe Asn GlyThr Ala Gly Val Trp Arg 225 230 235 240 Ile Lys Ala Leu Glu Glu Ser GlyGly Trp Met Glu Arg Thr Thr Val 245 250 255 Glu Asp Met Asp Ile Ala ValArg Ala His Leu Lys Gly Trp Lys Phe 260 265 270 Leu Phe Leu Asn Asp ValGlu Cys Gln Cys Glu Leu Pro Glu Ser Tyr 275 280 285 Glu Ala Tyr Arg LysGln Gln His Arg Trp His Ser Gly Pro Met Gln 290 295 300 Leu Phe Arg LeuCys Phe Val Asp Ile Ile Lys Ser Lys Ile Gly Phe 305 310 315 320 Trp LysLys Phe Asn Leu Ile Phe Leu Phe Phe Leu Leu Arg Lys Leu 325 330 335 IleLeu Pro Phe Tyr Ser Phe Thr Leu Phe Cys Ile Ile Leu Pro Met 340 345 350Thr Met Phe Val Pro Glu Ala Glu Leu Pro Asp Trp Val Val Cys Tyr 355 360365 Val Pro Ala Leu Met Ser Leu Leu Asn Ile Leu Pro Ser Pro Lys Ser 370375 380 Phe Pro Phe Ile Ile Pro Tyr Leu Leu Phe Glu Asn Thr Met Ser Val385 390 395 400 Thr Lys Phe Asn Ala Met Ile Ser Gly Leu Phe Gln Leu GlySer Ala 405 410 415 Tyr Glu Trp Val Val Thr Lys Lys Ser Gly Arg Ser SerGlu Gly Asp 420 425 430 Leu Ile Ala Leu Ala Pro Pro Lys Glu Pro Val LysHis Ala Thr Arg 435 440 445 Thr Gly Ser Ala Pro Asn Leu Asp Ala Val AlaLys Glu Glu Gln Gln 450 455 460 Gln Gln Gln Leu Ala Ala Ser Arg Lys AspAla Ala Ala Lys Lys Lys 465 470 475 480 Glu Lys His Asn Arg Ile Tyr LysLys Glu Leu Ala Leu Ser Met Leu 485 490 495 Leu Leu Thr Ala Ala Ala ArgSer Leu Leu Ser Lys His Gly Ile His 500 505 510 Phe Tyr Phe Leu Leu PheGln Gly Val Ser Phe Leu Leu Val Gly Leu 515 520 525 Asp Leu Ile Gly GluGln Val Glu 530 535 9 1221 DNA Zea mays 9 ccacgcgtcc gggcaggagctctgaagaaa ggaatggaat gtgactatgc atggcaaagc 60 gaatacattg ctatatttgatgctgatttc caacctgaac cagattttct gctccaaact 120 gtcccattcc ttctgcacaatccagaagtt gcacttgttc aagctcggtg gtccttcgtg 180 aatgacacga caagcctgctgacaagggta caaaagatgt tttacgacta ccacttcaaa 240 gttgaacaag aagcaggatcagcgaccttt gccttcttca gtttcaacgg aactgctgga 300 gtgtggcgta caggagccataagagatgca ggaggttgga aggaccgaac tacagttgaa 360 gacatggact tggcggttcgagcaacacta aagggctgga aattcgtata tgttggagac 420 gttagagtca agagtgaactgccgtccact tacaaggcct actgtcggca gcaattccgg 480 tggtctagtg gtggtgcaaacttattccgt aagatggcaa aggatgtttt gtttgccaag 540 gatatatcac tcgtcaagaagttctatatg ctctatagct tcttctttgt gaggagagtt 600 gtagcgccga cggctgcctgtattctctac aatgtcatca tccccatctc agtcacaatc 660 ccggagcttt acctaccagtgtggggtgtt gcctatattc ccatggtgct taccgtggtc 720 acagctataa gacatccaaaaaatctacac atactgccat tttggatttt gtttgagagt 780 gtgatgacat tgcatcggatgagggctgcg atgactggac tgctggagct agaaggattc 840 aaccagtgga ttgtgacaaagaaggtgggg aatgatctcg aggacactga agttcctttg 900 cttcagaaaa cccggaaaaggctgagagac agagtcaatc tccccgagat tggattttcg 960 gtgtttctct tcctctgtgcatcatacaac ctggtgttcc atgggaaaac aagctactac 1020 ttatatatgt accttcaggggttagcattt ctgttactag ggtttaactt cactggcaat 1080 tgttcttgct accaatgatagcatgtcaaa gctgtacgaa ttgctgattg atattcattt 1140 tctggtcatg cgttcgtawtgaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1200 aaaaaaaaaa aaaaaaaaaa g1221 10 354 PRT Zea mays 10 Met Glu Cys Asp Tyr Ala Trp Gln Ser Glu TyrIle Ala Ile Phe Asp 1 5 10 15 Ala Asp Phe Gln Pro Glu Pro Asp Phe LeuLeu Gln Thr Val Pro Phe 20 25 30 Leu Leu His Asn Pro Glu Val Ala Leu ValGln Ala Arg Trp Ser Phe 35 40 45 Val Asn Asp Thr Thr Ser Leu Leu Thr ArgVal Gln Lys Met Phe Tyr 50 55 60 Asp Tyr His Phe Lys Val Glu Gln Glu AlaGly Ser Ala Thr Phe Ala 65 70 75 80 Phe Phe Ser Phe Asn Gly Thr Ala GlyVal Trp Arg Thr Gly Ala Ile 85 90 95 Arg Asp Ala Gly Gly Trp Lys Asp ArgThr Thr Val Glu Asp Met Asp 100 105 110 Leu Ala Val Arg Ala Thr Leu LysGly Trp Lys Phe Val Tyr Val Gly 115 120 125 Asp Val Arg Val Lys Ser GluLeu Pro Ser Thr Tyr Lys Ala Tyr Cys 130 135 140 Arg Gln Gln Phe Arg TrpSer Ser Gly Gly Ala Asn Leu Phe Arg Lys 145 150 155 160 Met Ala Lys AspVal Leu Phe Ala Lys Asp Ile Ser Leu Val Lys Lys 165 170 175 Phe Tyr MetLeu Tyr Ser Phe Phe Phe Val Arg Arg Val Val Ala Pro 180 185 190 Thr AlaAla Cys Ile Leu Tyr Asn Val Ile Ile Pro Ile Ser Val Thr 195 200 205 IlePro Glu Leu Tyr Leu Pro Val Trp Gly Val Ala Tyr Ile Pro Met 210 215 220Val Leu Thr Val Val Thr Ala Ile Arg His Pro Lys Asn Leu His Ile 225 230235 240 Leu Pro Phe Trp Ile Leu Phe Glu Ser Val Met Thr Leu His Arg Met245 250 255 Arg Ala Ala Met Thr Gly Leu Leu Glu Leu Glu Gly Phe Asn GlnTrp 260 265 270 Ile Val Thr Lys Lys Val Gly Asn Asp Leu Glu Asp Thr GluVal Pro 275 280 285 Leu Leu Gln Lys Thr Arg Lys Arg Leu Arg Asp Arg ValAsn Leu Pro 290 295 300 Glu Ile Gly Phe Ser Val Phe Leu Phe Leu Cys AlaSer Tyr Asn Leu 305 310 315 320 Val Phe His Gly Lys Thr Ser Tyr Tyr LeuTyr Met Tyr Leu Gln Gly 325 330 335 Leu Ala Phe Leu Leu Leu Gly Phe AsnPhe Thr Gly Asn Cys Ser Cys 340 345 350 Tyr Gln 11 1065 DNA Zea mays 11atggaatgtg actatgcatg gcaaagcgaa tacattgcta tatttgatgc tgatttccaa 60cctgaaccag attttctgct ccaaactgtc ccattccttc tgcacaatcc agaagttgca 120cttgttcaag ctcggtggtc cttcgtgaat gacacgacaa gcctgctgac aagggtacaa 180aagatgtttt acgactacca cttcaaagtt gaacaagaag caggatcagc gacctttgcc 240ttcttcagtt tcaacggaac tgctggagtg tggcgtacag gagccataag agatgcagga 300ggttggaagg accgaactac agttgaagac atggacttgg cggttcgagc aacactaaag 360ggctggaaat tcgtatatgt tggagacgtt agagtcaaga gtgaactgcc gtccacttac 420aaggcctact gtcggcagca attccggtgg tctagtggtg gtgcaaactt attccgtaag 480atggcaaagg atgttttgtt tgccaaggat atatcactcg tcaagaagtt ctatatgctc 540tatagcttct tctttgtgag gagagttgta gcgccgacgg ctgcctgtat tctctacaat 600gtcatcatcc ccatctcagt cacaatcccg gagctttacc taccagtgtg gggtgttgcc 660tatattccca tggtgcttac cgtggtcaca gctataagac atccaaaaaa tctacacata 720ctgccatttt ggattttgtt tgagagtgtg atgacattgc atcggatgag ggctgcgatg 780actggactgc tggagctaga aggattcaac cagtggattg tgacaaagaa ggtggggaat 840gatctcgagg acactgaagt tcctttgctt cagaaaaccc ggaaaaggct gagagacaga 900gtcaatctcc ccgagattgg attttcggtg tttctcttcc tctgtgcatc atacaacctg 960gtgttccatg ggaaaacaag ctactactta tatatgtacc ttcaggggtt agcatttctg 1020ttactagggt ttaacttcac tggcaattgt tcttgctacc aatga 1065 12 354 PRT Zeamays 12 Met Glu Cys Asp Tyr Ala Trp Gln Ser Glu Tyr Ile Ala Ile Phe Asp1 5 10 15 Ala Asp Phe Gln Pro Glu Pro Asp Phe Leu Leu Gln Thr Val ProPhe 20 25 30 Leu Leu His Asn Pro Glu Val Ala Leu Val Gln Ala Arg Trp SerPhe 35 40 45 Val Asn Asp Thr Thr Ser Leu Leu Thr Arg Val Gln Lys Met PheTyr 50 55 60 Asp Tyr His Phe Lys Val Glu Gln Glu Ala Gly Ser Ala Thr PheAla 65 70 75 80 Phe Phe Ser Phe Asn Gly Thr Ala Gly Val Trp Arg Thr GlyAla Ile 85 90 95 Arg Asp Ala Gly Gly Trp Lys Asp Arg Thr Thr Val Glu AspMet Asp 100 105 110 Leu Ala Val Arg Ala Thr Leu Lys Gly Trp Lys Phe ValTyr Val Gly 115 120 125 Asp Val Arg Val Lys Ser Glu Leu Pro Ser Thr TyrLys Ala Tyr Cys 130 135 140 Arg Gln Gln Phe Arg Trp Ser Ser Gly Gly AlaAsn Leu Phe Arg Lys 145 150 155 160 Met Ala Lys Asp Val Leu Phe Ala LysAsp Ile Ser Leu Val Lys Lys 165 170 175 Phe Tyr Met Leu Tyr Ser Phe PhePhe Val Arg Arg Val Val Ala Pro 180 185 190 Thr Ala Ala Cys Ile Leu TyrAsn Val Ile Ile Pro Ile Ser Val Thr 195 200 205 Ile Pro Glu Leu Tyr LeuPro Val Trp Gly Val Ala Tyr Ile Pro Met 210 215 220 Val Leu Thr Val ValThr Ala Ile Arg His Pro Lys Asn Leu His Ile 225 230 235 240 Leu Pro PheTrp Ile Leu Phe Glu Ser Val Met Thr Leu His Arg Met 245 250 255 Arg AlaAla Met Thr Gly Leu Leu Glu Leu Glu Gly Phe Asn Gln Trp 260 265 270 IleVal Thr Lys Lys Val Gly Asn Asp Leu Glu Asp Thr Glu Val Pro 275 280 285Leu Leu Gln Lys Thr Arg Lys Arg Leu Arg Asp Arg Val Asn Leu Pro 290 295300 Glu Ile Gly Phe Ser Val Phe Leu Phe Leu Cys Ala Ser Tyr Asn Leu 305310 315 320 Val Phe His Gly Lys Thr Ser Tyr Tyr Leu Tyr Met Tyr Leu GlnGly 325 330 335 Leu Ala Phe Leu Leu Leu Gly Phe Asn Phe Thr Gly Asn CysSer Cys 340 345 350 Tyr Gln 13 1899 DNA Zea mays 13 gcctcttcgccgccgcctac gcggcctgga tgcgcgcccg cctcgactac ctcgcgccgc 60 cgctgcagttcctaaccaac gcctgcgtcc tcctcttcct ggtccagagc gtcgaccgcc 120 tcgtgctctgcctcggctgc ttctggatca agctcaaggg cgtcaggccc gtgccgccgc 180 tgcccgccgacaaggaggac gtcgaggccg gtcccgacgg cgtccccatg gtgctcgtgc 240 agatgcccatgtgcaatgag agagaggtgt accagcaatc catcggtgcg gtttgcagcc 300 tggactggccaaggtcaaat ttcctggtcc aggtgttgga tgactctgat gatgctacca 360 cttcggcacttatcaaggag gaggtggaga aatggcagcg agagggtgtg cgcatagtat 420 accggcaccgggtgatccgg gatggctaca aggctggaaa cctgaaatca gccatgaact 480 gcagttacgtgaaagactat gagttcgttg tcatcttcga tgctgatttc caaccacagg 540 cggacttcctgaagcgcacc gtgccccatt tcaagggaaa ggatgacgtc gggttggttc 600 aggcgagatggtcgttcgta aacaaggatg agaacttgct gaccaggctt cagaacataa 660 atctttgcttccacttcgag gtggagcagc aggtgaacgg ggcgtttctc aacttcttcg 720 ggttcaatggcaccgcggga gtctggagaa tcaaggcgct tgaggagtct ggaggatgga 780 tggagaggacgacggtggag gacatggaca tagctgttcg agcgcacctc aaagggtgga 840 agtttctctttctaaacgat gtcgagtgtc agtgtgaatt gccagaatcg tatgaagcgt 900 acagaaagcagcagcaccgg tggcactcag gtcccatgca attgtttagg ctctgctttg 960 tggatataatcaaatctaag atcggtttct ggaagaagtt caacctcata ttcctcttct 1020 tcctgctccggaagctgata ctacccttct actccttcac cctcttctgc gtgatcctcc 1080 ccatgacgatgttcgtcccc gaagccgagc tccccgcgtg ggtggtgtgc tacatcccgg 1140 cgacgatgtccatcctcaac atcctcccgt ccccgaaatc gttcccgttc atcgtcccgt 1200 acctgctgttcgagaacacc atgtcggtga ccaagttcaa cgccatggtc tccggcctgt 1260 tccagctggggagcgcctac gagtgggtcg tcaccaagaa gtcggggcgc tcctccgagg 1320 gcgacctcgtggccctcgtg gagaagcact ccaagcagca gagggtaggc tcggcgccca 1380 acctcgacgcgctgaccaag gagtcgaagg gcaccgagga ggagaagaat aagaagaaga 1440 ggaagaagaagcacaacagg atctacagga aggagctcgc gctgtccttc ctcctgctga 1500 ccgcggccgcccgcagcttg ctgtccgccc agggcgtcca cttctacttc ctcctgttcc 1560 agggggtttcgttcttggtc gtcgggctcg acctgatcgg cgagcaggtg gattgatagc 1620 agttgaataatgggttatat atatatatat atatatatat tttcgcttga agaaatcctc 1680 gcaggcatcatcaaattcaa agggctcttt gtgaagggaa gagcgtcgcc ttcttggatg 1740 cggaaaccttgggtccctgt tcctgttcca ggaggggtcg gaaacgtggg gagctgtgta 1800 gataggtatagcttggggtt tagcctgcga gatgcttttg ttcttccagg tttcgattct 1860 tttgtaagaatatttgtgcc cctacatgca agggctctt 1899 14 528 PRT Zea mays 14 Met Arg AlaArg Leu Asp Tyr Leu Ala Pro Pro Leu Gln Phe Leu Thr 1 5 10 15 Asn AlaCys Val Leu Leu Phe Leu Val Gln Ser Val Asp Arg Leu Val 20 25 30 Leu CysLeu Gly Cys Phe Trp Ile Lys Leu Lys Gly Val Arg Pro Val 35 40 45 Pro ProLeu Pro Ala Asp Lys Glu Asp Val Glu Ala Gly Pro Asp Gly 50 55 60 Val ProMet Val Leu Val Gln Met Pro Met Cys Asn Glu Arg Glu Val 65 70 75 80 TyrGln Gln Ser Ile Gly Ala Val Cys Ser Leu Asp Trp Pro Arg Ser 85 90 95 AsnPhe Leu Val Gln Val Leu Asp Asp Ser Asp Asp Ala Thr Thr Ser 100 105 110Ala Leu Ile Lys Glu Glu Val Glu Lys Trp Gln Arg Glu Gly Val Arg 115 120125 Ile Val Tyr Arg His Arg Val Ile Arg Asp Gly Tyr Lys Ala Gly Asn 130135 140 Leu Lys Ser Ala Met Asn Cys Ser Tyr Val Lys Asp Tyr Glu Phe Val145 150 155 160 Val Ile Phe Asp Ala Asp Phe Gln Pro Gln Ala Asp Phe LeuLys Arg 165 170 175 Thr Val Pro His Phe Lys Gly Lys Asp Asp Val Gly LeuVal Gln Ala 180 185 190 Arg Trp Ser Phe Val Asn Lys Asp Glu Asn Leu LeuThr Arg Leu Gln 195 200 205 Asn Ile Asn Leu Cys Phe His Phe Glu Val GluGln Gln Val Asn Gly 210 215 220 Ala Phe Leu Asn Phe Phe Gly Phe Asn GlyThr Ala Gly Val Trp Arg 225 230 235 240 Ile Lys Ala Leu Glu Glu Ser GlyGly Trp Met Glu Arg Thr Thr Val 245 250 255 Glu Asp Met Asp Ile Ala ValArg Ala His Leu Lys Gly Trp Lys Phe 260 265 270 Leu Phe Leu Asn Asp ValGlu Cys Gln Cys Glu Leu Pro Glu Ser Tyr 275 280 285 Glu Ala Tyr Arg LysGln Gln His Arg Trp His Ser Gly Pro Met Gln 290 295 300 Leu Phe Arg LeuCys Phe Val Asp Ile Ile Lys Ser Lys Ile Gly Phe 305 310 315 320 Trp LysLys Phe Asn Leu Ile Phe Leu Phe Phe Leu Leu Arg Lys Leu 325 330 335 IleLeu Pro Phe Tyr Ser Phe Thr Leu Phe Cys Val Ile Leu Pro Met 340 345 350Thr Met Phe Val Pro Glu Ala Glu Leu Pro Ala Trp Val Val Cys Tyr 355 360365 Ile Pro Ala Thr Met Ser Ile Leu Asn Ile Leu Pro Ser Pro Lys Ser 370375 380 Phe Pro Phe Ile Val Pro Tyr Leu Leu Phe Glu Asn Thr Met Ser Val385 390 395 400 Thr Lys Phe Asn Ala Met Val Ser Gly Leu Phe Gln Leu GlySer Ala 405 410 415 Tyr Glu Trp Val Val Thr Lys Lys Ser Gly Arg Ser SerGlu Gly Asp 420 425 430 Leu Val Ala Leu Val Glu Lys His Ser Lys Gln GlnArg Val Gly Ser 435 440 445 Ala Pro Asn Leu Asp Ala Leu Thr Lys Glu SerLys Gly Thr Glu Glu 450 455 460 Glu Lys Asn Lys Lys Lys Arg Lys Lys LysHis Asn Arg Ile Tyr Arg 465 470 475 480 Lys Glu Leu Ala Leu Ser Phe LeuLeu Leu Thr Ala Ala Ala Arg Ser 485 490 495 Leu Leu Ser Ala Gln Gly ValHis Phe Tyr Phe Leu Leu Phe Gln Gly 500 505 510 Val Ser Phe Leu Val ValGly Leu Asp Leu Ile Gly Glu Gln Val Asp 515 520 525 15 1587 DNA Zea mays15 atgcgcgccc gcctcgacta cctcgcgccg ccgctgcagt tcctaaccaa cgcctgcgtc 60ctcctcttcc tggtccagag cgtcgaccgc ctcgtgctct gcctcggctg cttctggatc 120aagctcaagg gcgtcaggcc cgtgccgccg ctgcccgccg acaaggagga cgtcgaggcc 180ggtcccgacg gcgtccccat ggtgctcgtg cagatgccca tgtgcaatga gagagaggtg 240taccagcaat ccatcggtgc ggtttgcagc ctggactggc caaggtcaaa tttcctggtc 300caggtgttgg atgactctga tgatgctacc acttcggcac ttatcaagga ggaggtggag 360aaatggcagc gagagggtgt gcgcatagta taccggcacc gggtgatccg ggatggctac 420aaggctggaa acctgaaatc agccatgaac tgcagttacg tgaaagacta tgagttcgtt 480gtcatcttcg atgctgattt ccaaccacag gcggacttcc tgaagcgcac cgtgccccat 540ttcaagggaa aggatgacgt cgggttggtt caggcgagat ggtcgttcgt aaacaaggat 600gagaacttgc tgaccaggct tcagaacata aatctttgct tccacttcga ggtggagcag 660caggtgaacg gggcgtttct caacttcttc gggttcaatg gcaccgcggg agtctggaga 720atcaaggcgc ttgaggagtc tggaggatgg atggagagga cgacggtgga ggacatggac 780atagctgttc gagcgcacct caaagggtgg aagtttctct ttctaaacga tgtcgagtgt 840cagtgtgaat tgccagaatc gtatgaagcg tacagaaagc agcagcaccg gtggcactca 900ggtcccatgc aattgtttag gctctgcttt gtggatataa tcaaatctaa gatcggtttc 960tggaagaagt tcaacctcat attcctcttc ttcctgctcc ggaagctgat actacccttc 1020tactccttca ccctcttctg cgtgatcctc cccatgacga tgttcgtccc cgaagccgag 1080ctccccgcgt gggtggtgtg ctacatcccg gcgacgatgt ccatcctcaa catcctcccg 1140tccccgaaat cgttcccgtt catcgtcccg tacctgctgt tcgagaacac catgtcggtg 1200accaagttca acgccatggt ctccggcctg ttccagctgg ggagcgccta cgagtgggtc 1260gtcaccaaga agtcggggcg ctcctccgag ggcgacctcg tggccctcgt ggagaagcac 1320tccaagcagc agagggtagg ctcggcgccc aacctcgacg cgctgaccaa ggagtcgaag 1380ggcaccgagg aggagaagaa taagaagaag aggaagaaga agcacaacag gatctacagg 1440aaggagctcg cgctgtcctt cctcctgctg accgcggccg cccgcagctt gctgtccgcc 1500cagggcgtcc acttctactt cctcctgttc cagggggttt cgttcttggt cgtcgggctc 1560gacctgatcg gcgagcaggt ggattga 1587 16 528 PRT Zea mays 16 Met Arg AlaArg Leu Asp Tyr Leu Ala Pro Pro Leu Gln Phe Leu Thr 1 5 10 15 Asn AlaCys Val Leu Leu Phe Leu Val Gln Ser Val Asp Arg Leu Val 20 25 30 Leu CysLeu Gly Cys Phe Trp Ile Lys Leu Lys Gly Val Arg Pro Val 35 40 45 Pro ProLeu Pro Ala Asp Lys Glu Asp Val Glu Ala Gly Pro Asp Gly 50 55 60 Val ProMet Val Leu Val Gln Met Pro Met Cys Asn Glu Arg Glu Val 65 70 75 80 TyrGln Gln Ser Ile Gly Ala Val Cys Ser Leu Asp Trp Pro Arg Ser 85 90 95 AsnPhe Leu Val Gln Val Leu Asp Asp Ser Asp Asp Ala Thr Thr Ser 100 105 110Ala Leu Ile Lys Glu Glu Val Glu Lys Trp Gln Arg Glu Gly Val Arg 115 120125 Ile Val Tyr Arg His Arg Val Ile Arg Asp Gly Tyr Lys Ala Gly Asn 130135 140 Leu Lys Ser Ala Met Asn Cys Ser Tyr Val Lys Asp Tyr Glu Phe Val145 150 155 160 Val Ile Phe Asp Ala Asp Phe Gln Pro Gln Ala Asp Phe LeuLys Arg 165 170 175 Thr Val Pro His Phe Lys Gly Lys Asp Asp Val Gly LeuVal Gln Ala 180 185 190 Arg Trp Ser Phe Val Asn Lys Asp Glu Asn Leu LeuThr Arg Leu Gln 195 200 205 Asn Ile Asn Leu Cys Phe His Phe Glu Val GluGln Gln Val Asn Gly 210 215 220 Ala Phe Leu Asn Phe Phe Gly Phe Asn GlyThr Ala Gly Val Trp Arg 225 230 235 240 Ile Lys Ala Leu Glu Glu Ser GlyGly Trp Met Glu Arg Thr Thr Val 245 250 255 Glu Asp Met Asp Ile Ala ValArg Ala His Leu Lys Gly Trp Lys Phe 260 265 270 Leu Phe Leu Asn Asp ValGlu Cys Gln Cys Glu Leu Pro Glu Ser Tyr 275 280 285 Glu Ala Tyr Arg LysGln Gln His Arg Trp His Ser Gly Pro Met Gln 290 295 300 Leu Phe Arg LeuCys Phe Val Asp Ile Ile Lys Ser Lys Ile Gly Phe 305 310 315 320 Trp LysLys Phe Asn Leu Ile Phe Leu Phe Phe Leu Leu Arg Lys Leu 325 330 335 IleLeu Pro Phe Tyr Ser Phe Thr Leu Phe Cys Val Ile Leu Pro Met 340 345 350Thr Met Phe Val Pro Glu Ala Glu Leu Pro Ala Trp Val Val Cys Tyr 355 360365 Ile Pro Ala Thr Met Ser Ile Leu Asn Ile Leu Pro Ser Pro Lys Ser 370375 380 Phe Pro Phe Ile Val Pro Tyr Leu Leu Phe Glu Asn Thr Met Ser Val385 390 395 400 Thr Lys Phe Asn Ala Met Val Ser Gly Leu Phe Gln Leu GlySer Ala 405 410 415 Tyr Glu Trp Val Val Thr Lys Lys Ser Gly Arg Ser SerGlu Gly Asp 420 425 430 Leu Val Ala Leu Val Glu Lys His Ser Lys Gln GlnArg Val Gly Ser 435 440 445 Ala Pro Asn Leu Asp Ala Leu Thr Lys Glu SerLys Gly Thr Glu Glu 450 455 460 Glu Lys Asn Lys Lys Lys Arg Lys Lys LysHis Asn Arg Ile Tyr Arg 465 470 475 480 Lys Glu Leu Ala Leu Ser Phe LeuLeu Leu Thr Ala Ala Ala Arg Ser 485 490 495 Leu Leu Ser Ala Gln Gly ValHis Phe Tyr Phe Leu Leu Phe Gln Gly 500 505 510 Val Ser Phe Leu Val ValGly Leu Asp Leu Ile Gly Glu Gln Val Asp 515 520 525 17 2551 DNA Zea mays17 cggacgcgtg ggtccggaga aagaacctcc actggtcaca gcaaacacta tcctgtccat 60ccttgctgct gactaccctg tggagaagct ttcttgctat gtttctgatg atggaggggc 120tctcctgact tttgaagcca tggctgaagc tgctagcttt gctaatatgt gggttccttt 180ctgtcgcaag cacaacattg agcctcgcaa tcctgacagc tacttcaatc ttaagaagga 240cccatacaag aacaaggttc gccaggattt tgtcaaggac aggaggaggg tcaagaggga 300gtatgacgag ttcaaggtca ggatcaatgg tctgcctgac tcgatacgcc gacgctctga 360tgcgtaccat gccagagagg aaatcaaggc tatgaagagg cagcgtgagg ccgctcttga 420tgatgcagtg gagcctgtta agatccctaa agctacatgg atggctgatg gcactcactg 480gcctggtact tggattcaac cttctgctga gcatacccgt ggtgatcatg ctggaattat 540tcaggtgatg ctgaaacctc ccagtgacga tcccttgtac ggcagcaccg gtgatgaagg 600cagacctctt gatttcaccg aggtcgacat ccgtttgcca atgctggtgt atgtgtcccg 660agagaagcgg cctggttatg atcacaacaa gaaggctgga gcgatgaatg ctctggtccg 720ttcatctgct gtcatgtcaa atggcccctt catcctcaac ctcgactgtg atcactatgt 780ctacaactcg caagctttcc gcgaagggat gtgcttcatg atggaccgtg gtggcgaccg 840cattggttat gtccagttcc cgcagcggtt tgagggcatc gatccatcag atcgctatgc 900caaccacaac accgtcttct tcgacgtcaa catgcgcgcg ctggatggtc tcatgggacc 960agtctatgtt ggcactggct gccttttccg ccgtgttgcc ctatatggat ttgaccctcc 1020gcgctccaag gagcacggtg gctgctgcag ctgttgcttc ccccagagac gcaagatcaa 1080agcttcagcc gctgcaccgg aggagacccg ggctctaagg atggcagact tcgacgagga 1140tgaaatgaac atgtcgtcgt tccccaagaa gtttggtaac tcgagcttcc tcatcgactc 1200cattccgatt gctgagttcc aagggcgccc gcttgctgat caccctggtg tcaagaacgg 1260ccgccctccc ggtgctctca ctgtcccccg tgaccttctg gatgcatcca cagtcgctga 1320ggccgtcagt gtcatctcat gctggtacga agacaagacc gagtggggcc accgtgttgg 1380ttggatctat ggctcggtga cggaggatgt ggtcactggg taccggatgc acaaccgggg 1440ttggaagtcg gtgtactgtg tcaccaagcg tgacgccttc cgcggcaccg cgcccatcaa 1500cctgaccgac cgtctccacc aggtgctccg gtgggctact ggatcagtgg agatcttctt 1560ctcccgcaac aacgcgctgc tggcgagccg cagaatgaag ttcttgcaga ggatcgcgta 1620cctgaacgtg ggtatctacc cgttcacgtc catcttcctg atcgtctact gcttcctgcc 1680ggcgctgtcg ctgttctcgg ggcagttcat cgtgaagacg ctgaacgtga cgttcctgac 1740gtacctgctg gtgatcacgc tgacgctgtg cctgctggcg gtgctggaga tcaagtggtc 1800ggggatcagc ctggaggagt ggtggcggaa cgagcagttc tggctgatcg gcggcacgag 1860cgcgcacctg gcggccgtgc tgcagggcct gctgaaggtg gtggcgggca tcgagatctc 1920cttcactctg acgtccaagt cgggcggcga cgacgtggac gacgagttcg cggacctgta 1980catcgtcaag tggacgtcgc tgatgatccc gcccatcgtg atcatgatgg tgaacctgat 2040cggcatcgcg gtcgggttca gccgcaccat ctacagcgag atcccgcagt ggagcaagct 2100gctgggcggc gtcttcttca gcttctgggt gctggcgcac ctgtacccgt tcgccaaggg 2160cctgatgggg cggaggggcc gcacgccgac catcgtcttc gtctgggcgg gcctcctctc 2220catcaccatc tcgctgctgt gggtggccat caacccgccg tcccagaacc agcagattgg 2280tgggtcgttc acattcccct gaaagctctc tgggccaatg gcggattcat gcatgcttcg 2340tcgcagtggg atccttgcgt tgctctgcat cagttcctgg ttgcagcggt tcactatctg 2400aggacgaagc tgctgggggg aatgtgggat cctgactgtc gaactggcta cttttttgtt 2460gtcgaagctg ataatactct tatgatgagc tatagtagta gttcttttgt tcttttgttt 2520ttgctatata taaaatacat gttctggtcc c 2551 18 720 PRT Zea mays 18 Met AlaGlu Ala Ala Ser Phe Ala Asn Met Trp Val Pro Phe Cys Arg 1 5 10 15 LysHis Asn Ile Glu Pro Arg Asn Pro Asp Ser Tyr Phe Asn Leu Lys 20 25 30 LysAsp Pro Tyr Lys Asn Lys Val Arg Gln Asp Phe Val Lys Asp Arg 35 40 45 ArgArg Val Lys Arg Glu Tyr Asp Glu Phe Lys Val Arg Ile Asn Gly 50 55 60 LeuPro Asp Ser Ile Arg Arg Arg Ser Asp Ala Tyr His Ala Arg Glu 65 70 75 80Glu Ile Lys Ala Met Lys Arg Gln Arg Glu Ala Ala Leu Asp Asp Ala 85 90 95Val Glu Pro Val Lys Ile Pro Lys Ala Thr Trp Met Ala Asp Gly Thr 100 105110 His Trp Pro Gly Thr Trp Ile Gln Pro Ser Ala Glu His Thr Arg Gly 115120 125 Asp His Ala Gly Ile Ile Gln Val Met Leu Lys Pro Pro Ser Asp Asp130 135 140 Pro Leu Tyr Gly Ser Thr Gly Asp Glu Gly Arg Pro Leu Asp PheThr 145 150 155 160 Glu Val Asp Ile Arg Leu Pro Met Leu Val Tyr Val SerArg Glu Lys 165 170 175 Arg Pro Gly Tyr Asp His Asn Lys Lys Ala Gly AlaMet Asn Ala Leu 180 185 190 Val Arg Ser Ser Ala Val Met Ser Asn Gly ProPhe Ile Leu Asn Leu 195 200 205 Asp Cys Asp His Tyr Val Tyr Asn Ser GlnAla Phe Arg Glu Gly Met 210 215 220 Cys Phe Met Met Asp Arg Gly Gly AspArg Ile Gly Tyr Val Gln Phe 225 230 235 240 Pro Gln Arg Phe Glu Gly IleAsp Pro Ser Asp Arg Tyr Ala Asn His 245 250 255 Asn Thr Val Phe Phe AspVal Asn Met Arg Ala Leu Asp Gly Leu Met 260 265 270 Gly Pro Val Tyr ValGly Thr Gly Cys Leu Phe Arg Arg Val Ala Leu 275 280 285 Tyr Gly Phe AspPro Pro Arg Ser Lys Glu His Gly Gly Cys Cys Ser 290 295 300 Cys Cys PhePro Gln Arg Arg Lys Ile Lys Ala Ser Ala Ala Ala Pro 305 310 315 320 GluGlu Thr Arg Ala Leu Arg Met Ala Asp Phe Asp Glu Asp Glu Met 325 330 335Asn Met Ser Ser Phe Pro Lys Lys Phe Gly Asn Ser Ser Phe Leu Ile 340 345350 Asp Ser Ile Pro Ile Ala Glu Phe Gln Gly Arg Pro Leu Ala Asp His 355360 365 Pro Gly Val Lys Asn Gly Arg Pro Pro Gly Ala Leu Thr Val Pro Arg370 375 380 Asp Leu Leu Asp Ala Ser Thr Val Ala Glu Ala Val Ser Val IleSer 385 390 395 400 Cys Trp Tyr Glu Asp Lys Thr Glu Trp Gly His Arg ValGly Trp Ile 405 410 415 Tyr Gly Ser Val Thr Glu Asp Val Val Thr Gly TyrArg Met His Asn 420 425 430 Arg Gly Trp Lys Ser Val Tyr Cys Val Thr LysArg Asp Ala Phe Arg 435 440 445 Gly Thr Ala Pro Ile Asn Leu Thr Asp ArgLeu His Gln Val Leu Arg 450 455 460 Trp Ala Thr Gly Ser Val Glu Ile PhePhe Ser Arg Asn Asn Ala Leu 465 470 475 480 Leu Ala Ser Arg Arg Met LysPhe Leu Gln Arg Ile Ala Tyr Leu Asn 485 490 495 Val Gly Ile Tyr Pro PheThr Ser Ile Phe Leu Ile Val Tyr Cys Phe 500 505 510 Leu Pro Ala Leu SerLeu Phe Ser Gly Gln Phe Ile Val Lys Thr Leu 515 520 525 Asn Val Thr PheLeu Thr Tyr Leu Leu Val Ile Thr Leu Thr Leu Cys 530 535 540 Leu Leu AlaVal Leu Glu Ile Lys Trp Ser Gly Ile Ser Leu Glu Glu 545 550 555 560 TrpTrp Arg Asn Glu Gln Phe Trp Leu Ile Gly Gly Thr Ser Ala His 565 570 575Leu Ala Ala Val Leu Gln Gly Leu Leu Lys Val Val Ala Gly Ile Glu 580 585590 Ile Ser Phe Thr Leu Thr Ser Lys Ser Gly Gly Asp Asp Val Asp Asp 595600 605 Glu Phe Ala Asp Leu Tyr Ile Val Lys Trp Thr Ser Leu Met Ile Pro610 615 620 Pro Ile Val Ile Met Met Val Asn Leu Ile Gly Ile Ala Val GlyPhe 625 630 635 640 Ser Arg Thr Ile Tyr Ser Glu Ile Pro Gln Trp Ser LysLeu Leu Gly 645 650 655 Gly Val Phe Phe Ser Phe Trp Val Leu Ala His LeuTyr Pro Phe Ala 660 665 670 Lys Gly Leu Met Gly Arg Arg Gly Arg Thr ProThr Ile Val Phe Val 675 680 685 Trp Ala Gly Leu Leu Ser Ile Thr Ile SerLeu Leu Trp Val Ala Ile 690 695 700 Asn Pro Pro Ser Gln Asn Gln Gln IleGly Gly Ser Phe Thr Phe Pro 705 710 715 720 19 1740 DNA Zea mays 19gagcagtgcc tgaacccaag tgcagtgcag cagccatggg agcagcagca gcaggtggcc 60acgcgctgcg cgccgtcggc gacgtcgtct cgttccccgc gaccgtcgcc gccttcgtgg 120aggcgctgct ccagggctgg gccgaggcca gggccgggct gctggtgccg ctgctccgcg 180ccgcggtgct gctgtgcacg gccatgtcgc tgatcgtgct ggccgagaag gtgttcctgg 240gcgcggtcag ctccgtggcg aagctgcggc gccggcgtcc ggggcgggtg tgcaggtgcg 300accccgacga ggaggcggct gcggcatccc aggcctatcc catggtgctc gtccagatcc 360ccatgtacaa cgagagggag gtttaccagc tatcaataga ggcagcctgc aggctcacat 420ggccggtaga tcgactaata gtgcaggtgc tggacgactc caccgactcc gtcatcaagg 480agctggtgaa gggcgagtgc gagcggtggg ccacggagga ggggatcaac gtcaagtacg 540agacgcgcaa ggacagggcc gggtacaagg ccggcaacct caaggagggg atgcgccacg 600cctacgtgcg cgcctgcgag ttcgtcgcca tgttcgacgc agacttccag ccgccgccgg 660acttcctcgt cagaaccgtc ccgttcctcg tccacaaccc cagcctcgcg ctcgtgcaga 720cacgctggaa gttcgtgaat gccaacgact gcttgctgac gagaatgcag gagatgtcca 780tggactacca tttcaaggtg gagcaggaag ctggctcttc cttatgcaac ttctttggat 840acaacggaac cgctggagta tggagaacgc aagcgatcgt cgagtccggg ggctgggagg 900accgaaccac tgctgaggac atggacttgg cgctgagagc agggctcctg ggctgggagt 960tcgtctacgt tggaagcata aaggttaaga gtgagctgcc gagcactctc aaggcgtacc 1020ggtcccagca gcaccgctgg tcatgcggac ctgccctcct gttcaagaaa atgttctggc 1080aaattctcgc tgccgagaga gtgtcggtct ggaagaagtg gtacatggtc tatgacttct 1140tcattgcccg gagaatcgta ggcaccttct acacgttctt ctttttcagc gtcctgattc 1200ctctgaacat tctgctaccc gaagcgcaga ttcctgtgtg ggagctcatc tatatcccca 1260tagctatcac tcttctcaac tctgttggga ctccaaggtc tatccatctg gtcatactgt 1320gggtcttgtt cgagaacgta atggcgttgc atcggtttaa agccatcttg atagggtttc 1380tcgaagctga cagggccaac gaatggatcg tgacgcaaaa gctggggaat ctgcagaagc 1440tgaaatcgat cgccagactt acaggaagct accgtttcaa agacaggttc catttcctgg 1500aggtgttcat tgggctgttc cttttggcct ctgcgtgctt tgactactta tacagagatg 1560actatgttta cctctttgtt cttccccaat cgatcatgta tttcgcgatt gggtttcagt 1620tcgttggtct caatgtctct gaagactgac caattgcaag acaactgaac gttttggttg 1680cgatattatg attgctcggg tcaaggttct tgtaaaaaaa aaaaaaaaaa aaaaaaaaaa 174020 537 PRT Zea mays 20 Met Gly Ala Ala Ala Ala Gly Gly His Ala Leu ArgAla Val Gly Asp 1 5 10 15 Val Val Ser Phe Pro Ala Thr Val Ala Ala PheVal Glu Ala Leu Leu 20 25 30 Gln Gly Trp Ala Glu Ala Arg Ala Gly Leu LeuVal Pro Leu Leu Arg 35 40 45 Ala Ala Val Leu Leu Cys Thr Ala Met Ser LeuIle Val Leu Ala Glu 50 55 60 Lys Val Phe Leu Gly Ala Val Ser Ser Val AlaLys Leu Arg Arg Arg 65 70 75 80 Arg Pro Gly Arg Val Cys Arg Cys Asp ProAsp Glu Glu Ala Ala Ala 85 90 95 Ala Ser Gln Ala Tyr Pro Met Val Leu ValGln Ile Pro Met Tyr Asn 100 105 110 Glu Arg Glu Val Tyr Gln Leu Ser IleGlu Ala Ala Cys Arg Leu Thr 115 120 125 Trp Pro Val Asp Arg Leu Ile ValGln Val Leu Asp Asp Ser Thr Asp 130 135 140 Ser Val Ile Lys Glu Leu ValLys Gly Glu Cys Glu Arg Trp Ala Thr 145 150 155 160 Glu Glu Gly Ile AsnVal Lys Tyr Glu Thr Arg Lys Asp Arg Ala Gly 165 170 175 Tyr Lys Ala GlyAsn Leu Lys Glu Gly Met Arg His Ala Tyr Val Arg 180 185 190 Ala Cys GluPhe Val Ala Met Phe Asp Ala Asp Phe Gln Pro Pro Pro 195 200 205 Asp PheLeu Val Arg Thr Val Pro Phe Leu Val His Asn Pro Ser Leu 210 215 220 AlaLeu Val Gln Thr Arg Trp Lys Phe Val Asn Ala Asn Asp Cys Leu 225 230 235240 Leu Thr Arg Met Gln Glu Met Ser Met Asp Tyr His Phe Lys Val Glu 245250 255 Gln Glu Ala Gly Ser Ser Leu Cys Asn Phe Phe Gly Tyr Asn Gly Thr260 265 270 Ala Gly Val Trp Arg Thr Gln Ala Ile Val Glu Ser Gly Gly TrpGlu 275 280 285 Asp Arg Thr Thr Ala Glu Asp Met Asp Leu Ala Leu Arg AlaGly Leu 290 295 300 Leu Gly Trp Glu Phe Val Tyr Val Gly Ser Ile Lys ValLys Ser Glu 305 310 315 320 Leu Pro Ser Thr Leu Lys Ala Tyr Arg Ser GlnGln His Arg Trp Ser 325 330 335 Cys Gly Pro Ala Leu Leu Phe Lys Lys MetPhe Trp Gln Ile Leu Ala 340 345 350 Ala Glu Arg Val Ser Val Trp Lys LysTrp Tyr Met Val Tyr Asp Phe 355 360 365 Phe Ile Ala Arg Arg Ile Val GlyThr Phe Tyr Thr Phe Phe Phe Phe 370 375 380 Ser Val Leu Ile Pro Leu AsnIle Leu Leu Pro Glu Ala Gln Ile Pro 385 390 395 400 Val Trp Glu Leu IleTyr Ile Pro Ile Ala Ile Thr Leu Leu Asn Ser 405 410 415 Val Gly Thr ProArg Ser Ile His Leu Val Ile Leu Trp Val Leu Phe 420 425 430 Glu Asn ValMet Ala Leu His Arg Phe Lys Ala Ile Leu Ile Gly Phe 435 440 445 Leu GluAla Asp Arg Ala Asn Glu Trp Ile Val Thr Gln Lys Leu Gly 450 455 460 AsnLeu Gln Lys Leu Lys Ser Ile Ala Arg Leu Thr Gly Ser Tyr Arg 465 470 475480 Phe Lys Asp Arg Phe His Phe Leu Glu Val Phe Ile Gly Leu Phe Leu 485490 495 Leu Ala Ser Ala Cys Phe Asp Tyr Leu Tyr Arg Asp Asp Tyr Val Tyr500 505 510 Leu Phe Val Leu Pro Gln Ser Ile Met Tyr Phe Ala Ile Gly PheGln 515 520 525 Phe Val Gly Leu Asn Val Ser Glu Asp 530 535 21 1834 DNAZea mays 21 aaccaccaca ccaccaccca atggaggccg gggaaatcgg cggggcccttgtcttcatcc 60 tcgccgccgc cgccgccgtc gcggccgccg tgtccgtcgg cgcggtcgacttcagccgcc 120 cgctcaccgc gggggcgccg ttcgacttcc aggcggcggt gtcctggctcatcggcatcc 180 tcgacggcac gtcctcggca gcggcggacg tggacggggc gtgggtggcggtgcgggccg 240 gggtgatcgc gccggtgctg caggtggcgg tgtgggcgtg catggtgatgtcggtgatgc 300 tggtggtgga ggccgtgtac aacagcgtca tcagcctcgg cgtcaaggccattgggtgga 360 ggcctgagtg gaggttcaag tggaagcccc tcgacagcgc cgacgaggagaaggggaccg 420 cccacttccc tatggtcctg gttcagatac ccatgtacaa cgagctggaggtgtacaagc 480 tgtcaatagc ggcagcatgt gagctgcagt ggccaaagga caggatagtaattcaagtgt 540 tggacgattc tactgacccc tttatcaaga atttggtgga gcttgaatgtgagcactggg 600 tgaacaaagg tgtcaatatt aagtatgcca caagaaccag ccgcaagggattcaaggcag 660 gagctctgaa gaaaggaatg gaatgtgact atgcatggca aagcgaatacattgctatat 720 ttgatgctga tttccaacct gaaccagatt ttctgctcca aactgtcccattccttctgc 780 acaatccaga agttgcactt gttcaagctc ggtggtcctt cgtgaatgacacgacaagcc 840 tgctgacaag ggtacaaaag atgttttacg actaccactt caaagttgaacaagaagcag 900 gatcagcgac ctttgccttc ttcagtttca acggaactgc tggagtgtggcgtacaggag 960 ccataagaga tgcaggaggt tggaaggacc gaactacagt tgaagacatggacttggcgg 1020 ttcgagcaac actaaagggc tggaaattcg tatatgttgg agacgttagagtcaagagtg 1080 aactgccgtc cacttacaag gcctgtcggc agcaattccg gtggtctagtggtggtgcaa 1140 acttattccg taagatggca aaggatgttt tgtttgccaa ggatatatcactcgtcaaga 1200 agttctatat gctctatagc ttcttctttg tgaggagagt tgtagcgccgacggctgcct 1260 gtattctcta caatgtcatc atccccatct cagtcacaat cccggagctttacctaccag 1320 tgtggggtgt tgcctatatt cccatggtgc ttaccgtggt cacagctataagacatccaa 1380 aaaatctaca catactgcca ttttggattt tgtttgagag tgtgatgacattgcatcgga 1440 tgagggctgc gatgactgga ctgctggagc tagaaggatt caaccagtggattgtgacaa 1500 agaaggtggg gaatgatctc gaggacactg aagttccttt gcttcagaaaacccggaaaa 1560 ggctgagaga cagagtcaat ctccccgaga ttggattttc ggtgtttctcttcctctgtg 1620 catcatacaa cctggtgttc catgggaaaa caagctacta cttatatatgtaccttcagg 1680 ggttagcatt tctgttacta gggtttaact tcactggcaa ttgttcttgctaccaatgat 1740 agcatgtcaa agctgtacga attgctgatt gatattcatt ttctggtcatgcgttcgtaa 1800 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa 1834 22 572 PRTZea mays 22 Met Glu Ala Gly Glu Ile Gly Gly Ala Leu Val Phe Ile Leu AlaAla 1 5 10 15 Ala Ala Ala Val Ala Ala Ala Val Ser Val Gly Ala Val AspPhe Ser 20 25 30 Arg Pro Leu Thr Ala Gly Ala Pro Phe Asp Phe Gln Ala AlaVal Ser 35 40 45 Trp Leu Ile Gly Ile Leu Asp Gly Thr Ser Ser Ala Ala AlaAsp Val 50 55 60 Asp Gly Ala Trp Val Ala Val Arg Ala Gly Val Ile Ala ProVal Leu 65 70 75 80 Gln Val Ala Val Trp Ala Cys Met Val Met Ser Val MetLeu Val Val 85 90 95 Glu Ala Val Tyr Asn Ser Val Ile Ser Leu Gly Val LysAla Ile Gly 100 105 110 Trp Arg Pro Glu Trp Arg Phe Lys Trp Lys Pro LeuAsp Ser Ala Asp 115 120 125 Glu Glu Lys Gly Thr Ala His Phe Pro Met ValLeu Val Gln Ile Pro 130 135 140 Met Tyr Asn Glu Leu Glu Val Tyr Lys LeuSer Ile Ala Ala Ala Cys 145 150 155 160 Glu Leu Gln Trp Pro Lys Asp ArgIle Val Ile Gln Val Leu Asp Asp 165 170 175 Ser Thr Asp Pro Phe Ile LysAsn Leu Val Glu Leu Glu Cys Glu His 180 185 190 Trp Val Asn Lys Gly ValAsn Ile Lys Tyr Ala Thr Arg Thr Ser Arg 195 200 205 Lys Gly Phe Lys AlaGly Ala Leu Lys Lys Gly Met Glu Cys Asp Tyr 210 215 220 Ala Trp Gln SerGlu Tyr Ile Ala Ile Phe Asp Ala Asp Phe Gln Pro 225 230 235 240 Glu ProAsp Phe Leu Leu Gln Thr Val Pro Phe Leu Leu His Asn Pro 245 250 255 GluVal Ala Leu Val Gln Ala Arg Trp Ser Phe Val Asn Asp Thr Thr 260 265 270Ser Leu Leu Thr Arg Val Gln Lys Met Phe Tyr Asp Tyr His Phe Lys 275 280285 Val Glu Gln Glu Ala Gly Ser Ala Thr Phe Ala Phe Phe Ser Phe Asn 290295 300 Gly Thr Ala Gly Val Trp Arg Thr Gly Ala Ile Arg Asp Ala Gly Gly305 310 315 320 Trp Lys Asp Arg Thr Thr Val Glu Asp Met Asp Leu Ala ValArg Ala 325 330 335 Thr Leu Lys Gly Trp Lys Phe Val Tyr Val Gly Asp ValArg Val Lys 340 345 350 Ser Glu Leu Pro Ser Thr Tyr Lys Ala Cys Arg GlnGln Phe Arg Trp 355 360 365 Ser Ser Gly Gly Ala Asn Leu Phe Arg Lys MetAla Lys Asp Val Leu 370 375 380 Phe Ala Lys Asp Ile Ser Leu Val Lys LysPhe Tyr Met Leu Tyr Ser 385 390 395 400 Phe Phe Phe Val Arg Arg Val ValAla Pro Thr Ala Ala Cys Ile Leu 405 410 415 Tyr Asn Val Ile Ile Pro IleSer Val Thr Ile Pro Glu Leu Tyr Leu 420 425 430 Pro Val Trp Gly Val AlaTyr Ile Pro Met Val Leu Thr Val Val Thr 435 440 445 Ala Ile Arg His ProLys Asn Leu His Ile Leu Pro Phe Trp Ile Leu 450 455 460 Phe Glu Ser ValMet Thr Leu His Arg Met Arg Ala Ala Met Thr Gly 465 470 475 480 Leu LeuGlu Leu Glu Gly Phe Asn Gln Trp Ile Val Thr Lys Lys Val 485 490 495 GlyAsn Asp Leu Glu Asp Thr Glu Val Pro Leu Leu Gln Lys Thr Arg 500 505 510Lys Arg Leu Arg Asp Arg Val Asn Leu Pro Glu Ile Gly Phe Ser Val 515 520525 Phe Leu Phe Leu Cys Ala Ser Tyr Asn Leu Val Phe His Gly Lys Thr 530535 540 Ser Tyr Tyr Leu Tyr Met Tyr Leu Gln Gly Leu Ala Phe Leu Leu Leu545 550 555 560 Gly Phe Asn Phe Thr Gly Asn Cys Ser Cys Tyr Gln 565 57023 2432 DNA Zea mays 23 ccacgcgtcc gccggtcctc ggctcatcag tattattattattattatta ttcggctcct 60 gctcatcagc tgcagcagtc gtgctccgga ccggagaagtcgaaatggag gagaggctgt 120 tcgccacgga gaagcacggt ggccgggcgc tctacaggctccacgccgtc acggtgttcc 180 tggggatatg cctgctgctc tgctacaggg cgacgcacgtcccggctgcc ggctccggcg 240 gcagggcggc gtggctgggg atgctcgcgg cggagctctggttcggcttc tactgggtca 300 tcacgcagtc cgtgcgctgg tgccccatcc gccgccgcaccttccacgac aggctcgccg 360 ccaggttcgg agagcggctc ccctgcgtgg acatcttcgtgtgcacagcg gacccgcggt 420 cggagccgcc gagccttgtc gtggccacgg tcctgtcggtgatggcgtac aactacccgc 480 ccgcgaagct caacgtctac ctctccgacg acggcggctccatcctcacc ttctacgctc 540 tgtgggaggc ctccgccttc gccaagcact ggctcccgttctgcaggagg tacggcgtcg 600 agccacggtc gccggccgct tacttcgccc agtctgatgagaagcctcgt catgatccgc 660 cgcacgcctt gcaggagtgg acgtccgtca aaaacctatacgatgaaatg acggagcgga 720 ttgactccgc tgctcggacg ggcaatgttc ctgaagaaactagagcgaaa cacaaagggt 780 tttctgagtg ggatacgggt attacctcaa aagaccaccacccgatcgtt cagattctga 840 tagatgggaa agacaaggct gtagctgaca acgaaggcaatgtgctgccg acgctggtgt 900 acgtggcacg agagaagagg cctcagtacc accacaacttcaaagccggg gcgatgaacg 960 ctctgatccg agtatcgtcc gtgataagca acagccctatcatcctgaac gtggactgcg 1020 acatgtattc caacaacagc gacacgatca gagacgcgctgtgcttcttc ctcgacgaag 1080 aaacgggcca caggatcgcg ttcgtgcagt accctcagaactacaacaac ctcaccaaga 1140 acaacatata cggcaactcc ctcaatgtca tcaaccaggtggagctgagc ggcctggacg 1200 cttggggcgg cccgctgtac atcggcacgg gatgcttccataggagggag accctgtgcg 1260 gcaggaggtt caccgaggac tacaaggaag actgggacagaggaaccaag gagcagcagc 1320 agcaccgcca ccgcgtcgac ggcgagaccg aagcgaaggccaagtcgcta gcgacctgcg 1380 cctacgagca cgacgacgac acgcggtggg gagacgaggtggggctcaag tacggctgct 1440 cggtggagga cgtcatcacg gggctggcga tacactgcagagggtgggag tcggtgtaca 1500 gcaaccccgc gagagcggcg ttcgtcggcg tcgcgccgaccacgctcgcc cagaccatac 1560 tgcagcacaa gcggtggagc gagggcaact tcggcatcttcgtttccagg tactgcccct 1620 tcgtctttgg acgacggggc aaaaccaggt tgccgcaccagatgggctac tccatctacg 1680 ggctatgggc gcccaactcg ctgcctacgc tgtactacgctgtcgtccct tcgctgtgcc 1740 tgctcaaggg cacccctctg ttccctgagc tcacgagtccgtggatcgcg cctttcgtct 1800 acgtcgcggt cgccaagaac gtctacagcg cgtgggaggcgctgtggtgc ggagacacgc 1860 tgagagggtg gtggaacggg cagaggatgt ggctggtccggagaacgacc tcgtacctct 1920 acggcttcgt cgacaccgtc agggactcgc tggggctgtccaagatgggc ttcgtggtgt 1980 cgtccaaggt gagcgacgag gacgaggcca agaggtacgagcaggagatg atggagttcg 2040 ggacggcgtc gccggagtac gtgatcgtcg cggccgtcgcgctgctcaac ctcgtgtgcc 2100 tggcagggat ggcggcggca ctggatgtgt tcttcgtccaggtcgctctc tgcggggtgc 2160 tggtgctcct caacgtcccg gtctatgaag ccatgttcgtcaggaaggac agggggagga 2220 tgccgttccc gatcacgcta gcctccgttg gctttgtgacgctggccctc attgtgccat 2280 tcttttgact ttgaggtgct aataatacgt gtacgggcacacgcacgttc gcatgtatga 2340 cgattatggg caacaggcgt gtaataccac taatacctattaaacactcc agtctccaag 2400 tgatccattg ctacaaaaaa aaaaaaaaaa aa 2432 24727 PRT Zea mays 24 Met Glu Glu Arg Leu Phe Ala Thr Glu Lys His Gly GlyArg Ala Leu 1 5 10 15 Tyr Arg Leu His Ala Val Thr Val Phe Leu Gly IleCys Leu Leu Leu 20 25 30 Cys Tyr Arg Ala Thr His Val Pro Ala Ala Gly SerGly Gly Arg Ala 35 40 45 Ala Trp Leu Gly Met Leu Ala Ala Glu Leu Trp PheGly Phe Tyr Trp 50 55 60 Val Ile Thr Gln Ser Val Arg Trp Cys Pro Ile ArgArg Arg Thr Phe 65 70 75 80 His Asp Arg Leu Ala Ala Arg Phe Gly Glu ArgLeu Pro Cys Val Asp 85 90 95 Ile Phe Val Cys Thr Ala Asp Pro Arg Ser GluPro Pro Ser Leu Val 100 105 110 Val Ala Thr Val Leu Ser Val Met Ala TyrAsn Tyr Pro Pro Ala Lys 115 120 125 Leu Asn Val Tyr Leu Ser Asp Asp GlyGly Ser Ile Leu Thr Phe Tyr 130 135 140 Ala Leu Trp Glu Ala Ser Ala PheAla Lys His Trp Leu Pro Phe Cys 145 150 155 160 Arg Arg Tyr Gly Val GluPro Arg Ser Pro Ala Ala Tyr Phe Ala Gln 165 170 175 Ser Asp Glu Lys ProArg His Asp Pro Pro His Ala Leu Gln Glu Trp 180 185 190 Thr Ser Val LysAsn Leu Tyr Asp Glu Met Thr Glu Arg Ile Asp Ser 195 200 205 Ala Ala ArgThr Gly Asn Val Pro Glu Glu Thr Arg Ala Lys His Lys 210 215 220 Gly PheSer Glu Trp Asp Thr Gly Ile Thr Ser Lys Asp His His Pro 225 230 235 240Ile Val Gln Ile Leu Ile Asp Gly Lys Asp Lys Ala Val Ala Asp Asn 245 250255 Glu Gly Asn Val Leu Pro Thr Leu Val Tyr Val Ala Arg Glu Lys Arg 260265 270 Pro Gln Tyr His His Asn Phe Lys Ala Gly Ala Met Asn Ala Leu Ile275 280 285 Arg Val Ser Ser Val Ile Ser Asn Ser Pro Ile Ile Leu Asn ValAsp 290 295 300 Cys Asp Met Tyr Ser Asn Asn Ser Asp Thr Ile Arg Asp AlaLeu Cys 305 310 315 320 Phe Phe Leu Asp Glu Glu Thr Gly His Arg Ile AlaPhe Val Gln Tyr 325 330 335 Pro Gln Asn Tyr Asn Asn Leu Thr Lys Asn AsnIle Tyr Gly Asn Ser 340 345 350 Leu Asn Val Ile Asn Gln Val Glu Leu SerGly Leu Asp Ala Trp Gly 355 360 365 Gly Pro Leu Tyr Ile Gly Thr Gly CysPhe His Arg Arg Glu Thr Leu 370 375 380 Cys Gly Arg Arg Phe Thr Glu AspTyr Lys Glu Asp Trp Asp Arg Gly 385 390 395 400 Thr Lys Glu Gln Gln GlnHis Arg His Arg Val Asp Gly Glu Thr Glu 405 410 415 Ala Lys Ala Lys SerLeu Ala Thr Cys Ala Tyr Glu His Asp Asp Asp 420 425 430 Thr Arg Trp GlyAsp Glu Val Gly Leu Lys Tyr Gly Cys Ser Val Glu 435 440 445 Asp Val IleThr Gly Leu Ala Ile His Cys Arg Gly Trp Glu Ser Val 450 455 460 Tyr SerAsn Pro Ala Arg Ala Ala Phe Val Gly Val Ala Pro Thr Thr 465 470 475 480Leu Ala Gln Thr Ile Leu Gln His Lys Arg Trp Ser Glu Gly Asn Phe 485 490495 Gly Ile Phe Val Ser Arg Tyr Cys Pro Phe Val Phe Gly Arg Arg Gly 500505 510 Lys Thr Arg Leu Pro His Gln Met Gly Tyr Ser Ile Tyr Gly Leu Trp515 520 525 Ala Pro Asn Ser Leu Pro Thr Leu Tyr Tyr Ala Val Val Pro SerLeu 530 535 540 Cys Leu Leu Lys Gly Thr Pro Leu Phe Pro Glu Leu Thr SerPro Trp 545 550 555 560 Ile Ala Pro Phe Val Tyr Val Ala Val Ala Lys AsnVal Tyr Ser Ala 565 570 575 Trp Glu Ala Leu Trp Cys Gly Asp Thr Leu ArgGly Trp Trp Asn Gly 580 585 590 Gln Arg Met Trp Leu Val Arg Arg Thr ThrSer Tyr Leu Tyr Gly Phe 595 600 605 Val Asp Thr Val Arg Asp Ser Leu GlyLeu Ser Lys Met Gly Phe Val 610 615 620 Val Ser Ser Lys Val Ser Asp GluAsp Glu Ala Lys Arg Tyr Glu Gln 625 630 635 640 Glu Met Met Glu Phe GlyThr Ala Ser Pro Glu Tyr Val Ile Val Ala 645 650 655 Ala Val Ala Leu LeuAsn Leu Val Cys Leu Ala Gly Met Ala Ala Ala 660 665 670 Leu Asp Val PhePhe Val Gln Val Ala Leu Cys Gly Val Leu Val Leu 675 680 685 Leu Asn ValPro Val Tyr Glu Ala Met Phe Val Arg Lys Asp Arg Gly 690 695 700 Arg MetPro Phe Pro Ile Thr Leu Ala Ser Val Gly Phe Val Thr Leu 705 710 715 720Ala Leu Ile Val Pro Phe Phe 725 25 1190 DNA Zea mays 25 gcacgagcacacgcacgcat acacagcaca gagtgaggta agcatccgaa aaaagctgtg 60 atctgatcgacatggccgcc gccaccatgg ctctcacctc ccgcgcgctc gtcggcaagc 120 cggcgaccagcaccagggac gtcttcggcg aggggcgcat caccatgcgc aagactgctg 180 gcaagcccaagccagcggcg tccggcagcc cctggtacgg ggccgaccgc gtcctgtacc 240 tgggcccgctgtccggccag cccccaagct acctgaccgg cgagttcccc ggcgactacg 300 gctgggacaccgcggggctg tccgccgacc cggagacttt cgccaagaac cgcgagctgg 360 aggtgatccactgccgctgg gccatgctgg gcgcgctcgg gtgcgtgttc ccggagctgc 420 tcgcccgcaacggcgtcaag ttcggcgagg ccgtgtggtt caaggccggg tcccagatct 480 tcagcgagggcgggcttgac tacctcggca acccgagcct gatccacgcg cagagcatcc 540 tggccatctgggcgtgccag gtggtgctca tgggcgccgt cgaggggtac cgcatcgccg 600 gtggcccgctcggggaggtg gtcgacccgc tgtaccccgg cggcagcttc gacccgctcg 660 ggctcgccgacgacccggag gcgttcgcgg agctcaaggt caaggagatc aagaacggcc 720 gcctcgccatgttctccatg ttcggcttct tcgtgcaggc catcgtcacc ggcaagggtc 780 ccgttgagaacctcgccgac cacctcgctg accctgtcaa caacaacgcc tgggcctacg 840 ctaccaacttcgtccccggc aagtgagcga gggcatatac atacttgtat gcgtgtaccg 900 tagacatacgtattcgtata tgtactgcag gagatgtacc agtatttgtg aagaagggag 960 catgggctcagagattgtac cagtagtgta cgtatttgca tgcatgcttt ggaaaaaaaa 1020 aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa cctcgaattt gccaccttct 1080 tgctgttttaaaaatatttt atcacttata tgctgcatta ggtggtagga gttgaatggt 1140 taaacctttgctgcataact tccttgaatt attgagtatc aaggatgata 1190 26 264 PRT Zea mays 26Met Ala Ala Ala Thr Met Ala Leu Thr Ser Arg Ala Leu Val Gly Lys 1 5 1015 Pro Ala Thr Ser Thr Arg Asp Val Phe Gly Glu Gly Arg Ile Thr Met 20 2530 Arg Lys Thr Ala Gly Lys Pro Lys Pro Ala Ala Ser Gly Ser Pro Trp 35 4045 Tyr Gly Ala Asp Arg Val Leu Tyr Leu Gly Pro Leu Ser Gly Gln Pro 50 5560 Pro Ser Tyr Leu Thr Gly Glu Phe Pro Gly Asp Tyr Gly Trp Asp Thr 65 7075 80 Ala Gly Leu Ser Ala Asp Pro Glu Thr Phe Ala Lys Asn Arg Glu Leu 8590 95 Glu Val Ile His Cys Arg Trp Ala Met Leu Gly Ala Leu Gly Cys Val100 105 110 Phe Pro Glu Leu Leu Ala Arg Asn Gly Val Lys Phe Gly Glu AlaVal 115 120 125 Trp Phe Lys Ala Gly Ser Gln Ile Phe Ser Glu Gly Gly LeuAsp Tyr 130 135 140 Leu Gly Asn Pro Ser Leu Ile His Ala Gln Ser Ile LeuAla Ile Trp 145 150 155 160 Ala Cys Gln Val Val Leu Met Gly Ala Val GluGly Tyr Arg Ile Ala 165 170 175 Gly Gly Pro Leu Gly Glu Val Val Asp ProLeu Tyr Pro Gly Gly Ser 180 185 190 Phe Asp Pro Leu Gly Leu Ala Asp AspPro Glu Ala Phe Ala Glu Leu 195 200 205 Lys Val Lys Glu Ile Lys Asn GlyArg Leu Ala Met Phe Ser Met Phe 210 215 220 Gly Phe Phe Val Gln Ala IleVal Thr Gly Lys Gly Pro Val Glu Asn 225 230 235 240 Leu Ala Asp His LeuAla Asp Pro Val Asn Asn Asn Ala Trp Ala Tyr 245 250 255 Ala Thr Asn PheVal Pro Gly Lys 260 27 2351 DNA Zea mays 27 gaggaaggga tggccgggagcagcgtccgc ggcggcagca actgcccacc gctgttcgtg 60 acggagaaac caacgcggatggcgaggtac gcttaccggc tgttcgcgag cacggtcctc 120 gcgggggttc ttctggtatggctgtacaga gcaacgcacg tgccgccgat gagcagcggc 180 gcccggtggt gggcgtggcttgggctctcc gcggcggagc tctggttcgg cttctactgg 240 gtgctgacgc tgtccgtgcggtggagcccc gtcttccgcc gcgccttccc ggaccagctc 300 ttgcgaaggt acaaggaagagcagcttcct ggggtggaca tatttgtgtg tacagcagac 360 cccactgttg agccgccaatgcttgtcatc tccactgtcc tatctgttat ggcttatgac 420 tacccgaagg agaagttgaacatatatttg tctgatgatg ctggttccat cataacattg 480 tatgctctat atgaagcatcagagtttgca aagcactggc ttccattttg caataagtac 540 caagtggagc ccaggtcaccagctgcctac tttggtacag aagctagccc tccagatgca 600 tgtgaccgta aagagtggttttctttgaag gagatgcaca aagatttggc agctcgagtg 660 aattcagttg ttaattcagggaagatccct gaagtttcaa aatgcaagct tatgggcttc 720 tccaggtgga gcgagaatgcaagttttaga gatcaccctt caatagttca gattttaatt 780 gatggaaaca aaaggaaagcaactgatatc gatggaaaag tgttgcccac actggtttat 840 atggctcgtg agaagagacctcaagaacat catcacttca aagctggatc actgaatgct 900 ttgataaggg tatcatcggtgataagcaac agcccagtca ttatgaatgt ggactgtgat 960 atgtactcca acaattcagggtctatcaga gatgcattgt gcttcttcca agacgaacag 1020 ctaggtcaag atattgcttttgttcagtat cctcagaact tcgaaaatgt ggtgcaaaat 1080 gacatctatg gcaatcccatcaacaccgtc aatgagttgg accatccttg cttggatgga 1140 tggggtggaa tgtgttactatggcacagga tgcttccatc ggagagaggc tctatgtggg 1200 cgaatataca gtccagactacaaggaagat tggactaggg tggcgaggaa aactgaagat 1260 gtcattgact tggaaggaatggctgagtca cttgtgactt gcacatatga gcacaacacc 1320 ctttggggag tcgagaagggagttatatat ggttgcccac tggaggatgt cattacagga 1380 ttgcagatcc agtgccgtgggtggagatca gtttaccaca acccgccaag aaaggggttt 1440 ttaggcatgg cccctacctcactaggacag attctggttc agcacaagag atggacagaa 1500 gggttcctcc agatctccctctcaaagtac agcccgtttc tgctaggtca caggaagatc 1560 agcctgggcc ttcaaatgggttactccgtc tgcgggttct gggctgctaa cagcttcccc 1620 accctttact atgtcactatcccttcactt tgcttcctca atggcatctc attgttccct 1680 gagataacca gtccctggtttgtaccgttt gcatacgttg ctgtggctgc atactcctgc 1740 agcttggtgg agtccctgcaatgtggcgac actgctgttg agtggtggaa cgcgcaaagg 1800 atgtggcttt tcagaagaatcacctcatac ctcttggcag ccatcgacac aatccgcaga 1860 atgcttggcg tcaccgagtcggggttcacc ctgacgacga aggtgaccga tccgcaggcc 1920 ctagagaggt acaagaaggggatgatggag tttgggtcct tctccgcgat gtttgcgatc 1980 attacaaccg ttgcactgcttaacctggcg tgcatgatgc tcggggtggc aaaggttttg 2040 ttgcgtaaag gagcggtgatgagtctggga gctatgtttg tgcaggccgt tctatgtgcg 2100 ctgatagtag cgatcaatttcccagtgtat gaagcaatgt tcgcccgcaa ggacagtggc 2160 agattaccag cttctgtcggtgtagtttcg ctctgcattg tattgccatt ctgtatactt 2220 ccaaccaact tgtagatgtggagctggtga aagatgatat atatatttga aacccgatgg 2280 tgaaagttat aagaactgtactgatataat atatttccaa gaaaatataa aaatctaaaa 2340 aaaaaaaaaa a 2351 28741 PRT Zea mays 28 Met Ala Gly Ser Ser Val Arg Gly Gly Ser Asn Cys ProPro Leu Phe 1 5 10 15 Val Thr Glu Lys Pro Thr Arg Met Ala Arg Tyr AlaTyr Arg Leu Phe 20 25 30 Ala Ser Thr Val Leu Ala Gly Val Leu Leu Val TrpLeu Tyr Arg Ala 35 40 45 Thr His Val Pro Pro Met Ser Ser Gly Ala Arg TrpTrp Ala Trp Leu 50 55 60 Gly Leu Ser Ala Ala Glu Leu Trp Phe Gly Phe TyrTrp Val Leu Thr 65 70 75 80 Leu Ser Val Arg Trp Ser Pro Val Phe Arg ArgAla Phe Pro Asp Gln 85 90 95 Leu Leu Arg Arg Tyr Lys Glu Glu Gln Leu ProGly Val Asp Ile Phe 100 105 110 Val Cys Thr Ala Asp Pro Thr Val Glu ProPro Met Leu Val Ile Ser 115 120 125 Thr Val Leu Ser Val Met Ala Tyr AspTyr Pro Lys Glu Lys Leu Asn 130 135 140 Ile Tyr Leu Ser Asp Asp Ala GlySer Ile Ile Thr Leu Tyr Ala Leu 145 150 155 160 Tyr Glu Ala Ser Glu PheAla Lys His Trp Leu Pro Phe Cys Asn Lys 165 170 175 Tyr Gln Val Glu ProArg Ser Pro Ala Ala Tyr Phe Gly Thr Glu Ala 180 185 190 Ser Pro Pro AspAla Cys Asp Arg Lys Glu Trp Phe Ser Leu Lys Glu 195 200 205 Met His LysAsp Leu Ala Ala Arg Val Asn Ser Val Val Asn Ser Gly 210 215 220 Lys IlePro Glu Val Ser Lys Cys Lys Leu Met Gly Phe Ser Arg Trp 225 230 235 240Ser Glu Asn Ala Ser Phe Arg Asp His Pro Ser Ile Val Gln Ile Leu 245 250255 Ile Asp Gly Asn Lys Arg Lys Ala Thr Asp Ile Asp Gly Lys Val Leu 260265 270 Pro Thr Leu Val Tyr Met Ala Arg Glu Lys Arg Pro Gln Glu His His275 280 285 His Phe Lys Ala Gly Ser Leu Asn Ala Leu Ile Arg Val Ser SerVal 290 295 300 Ile Ser Asn Ser Pro Val Ile Met Asn Val Asp Cys Asp MetTyr Ser 305 310 315 320 Asn Asn Ser Gly Ser Ile Arg Asp Ala Leu Cys PhePhe Gln Asp Glu 325 330 335 Gln Leu Gly Gln Asp Ile Ala Phe Val Gln TyrPro Gln Asn Phe Glu 340 345 350 Asn Val Val Gln Asn Asp Ile Tyr Gly AsnPro Ile Asn Thr Val Asn 355 360 365 Glu Leu Asp His Pro Cys Leu Asp GlyTrp Gly Gly Met Cys Tyr Tyr 370 375 380 Gly Thr Gly Cys Phe His Arg ArgGlu Ala Leu Cys Gly Arg Ile Tyr 385 390 395 400 Ser Pro Asp Tyr Lys GluAsp Trp Thr Arg Val Ala Arg Lys Thr Glu 405 410 415 Asp Val Ile Asp LeuGlu Gly Met Ala Glu Ser Leu Val Thr Cys Thr 420 425 430 Tyr Glu His AsnThr Leu Trp Gly Val Glu Lys Gly Val Ile Tyr Gly 435 440 445 Cys Pro LeuGlu Asp Val Ile Thr Gly Leu Gln Ile Gln Cys Arg Gly 450 455 460 Trp ArgSer Val Tyr His Asn Pro Pro Arg Lys Gly Phe Leu Gly Met 465 470 475 480Ala Pro Thr Ser Leu Gly Gln Ile Leu Val Gln His Lys Arg Trp Thr 485 490495 Glu Gly Phe Leu Gln Ile Ser Leu Ser Lys Tyr Ser Pro Phe Leu Leu 500505 510 Gly His Arg Lys Ile Ser Leu Gly Leu Gln Met Gly Tyr Ser Val Cys515 520 525 Gly Phe Trp Ala Ala Asn Ser Phe Pro Thr Leu Tyr Tyr Val ThrIle 530 535 540 Pro Ser Leu Cys Phe Leu Asn Gly Ile Ser Leu Phe Pro GluIle Thr 545 550 555 560 Ser Pro Trp Phe Val Pro Phe Ala Tyr Val Ala ValAla Ala Tyr Ser 565 570 575 Cys Ser Leu Val Glu Ser Leu Gln Cys Gly AspThr Ala Val Glu Trp 580 585 590 Trp Asn Ala Gln Arg Met Trp Leu Phe ArgArg Ile Thr Ser Tyr Leu 595 600 605 Leu Ala Ala Ile Asp Thr Ile Arg ArgMet Leu Gly Val Thr Glu Ser 610 615 620 Gly Phe Thr Leu Thr Thr Lys ValThr Asp Pro Gln Ala Leu Glu Arg 625 630 635 640 Tyr Lys Lys Gly Met MetGlu Phe Gly Ser Phe Ser Ala Met Phe Ala 645 650 655 Ile Ile Thr Thr ValAla Leu Leu Asn Leu Ala Cys Met Met Leu Gly 660 665 670 Val Ala Lys ValLeu Leu Arg Lys Gly Ala Val Met Ser Leu Gly Ala 675 680 685 Met Phe ValGln Ala Val Leu Cys Ala Leu Ile Val Ala Ile Asn Phe 690 695 700 Pro ValTyr Glu Ala Met Phe Ala Arg Lys Asp Ser Gly Arg Leu Pro 705 710 715 720Ala Ser Val Gly Val Val Ser Leu Cys Ile Val Leu Pro Phe Cys Ile 725 730735 Leu Pro Thr Asn Leu 740 29 2318 DNA Zea mays 29 tctagacgcacacagacaga aagagtcggt acaaatcgtc gagggaggcc gggcgcgcgt 60 aggaacagaaagacacgcag ccagattgac agagctgagt gtgagtacgt actacgtaga 120 tacctacagctatactgtac tgtacagagt gggaggaaaa gggagggaga gagagggacg 180 tgtacacacacgcgcgtaca aaaatacaga ggaagcttag cttgcctcca accccgaccg 240 acactcgggctcgtgaaccc cgtcccgtaa tttctctata tatcccgtcc ctccccccac 300 gtacttcactcggcctccat ctccccacgc accccggcgc ccgcgccgcg ccaccttctc 360 tcccccctcctcctcctcct ctctctctct ctctctctct gccacacagc acaccagaag 420 ggcagcaggggaggtagaga gaggtagctt cgcattctcg gtttccctcc gcgcgtgtcc 480 tcccagcctcgacagagaga agggccacca tcgtccctgc ctgattgcgc gccaggcagg 540 caggcattatggcgccgggc ggccggcgca gcaacggcga gacgccgaca ggacagcagc 600 agcagcagcagcaggctgac ggcaggcgcg ggtgcgcatg cggcgggttc cccgtgtgcg 660 cgtgcgccggcgcggcggcg gtggcgtccg ccgcctcctc cgccgacatg gaccgcgtgg 720 cggtggccgccaccgagggc cagatcggcg ccgtcaacga cgagagctgg gtggcggtcg 780 acctcagcgacgacggcctc tcctccgccg ccgacccggg ggccgtcgcg ctcgaggaac 840 gccccgtcttccgcaccgag aagatcaagg gtgtcctcct ccacccctac agggtgctca 900 tcttcgtgcgcctgatcgcg ttcacgctgt tcgtgatctg gcgcatctcg caccgcaacc 960 cggacgcgctgtggctgtgg gtgacgtcga tcgcgggcga gttctggttc ggcttctcgt 1020 ggctgctggaccagctgccg aagctgaacc cgatcaaccg cgtgccggac ctgggggcgc 1080 tgcggcagcggttcgaccgc gccgacggga cgtcgcggct gccggggctg gacatcttcg 1140 tgaccacggcggacccgttc aaggagccga tcctgagcac ggccaactcc atcctctcca 1200 tcctggccgccgactacccc gtggagcgca acacgtgcta cctctccgac gactcgggca 1260 tgctgctcacgtacgaggcc atggcggagg ccgccaagtt cgccaccgtc tgggtgccct 1320 tctgccgcaagcacggcatc gagccgcgcg gccccgagag ctacttcgag ctcaagtccc 1380 acccctacatgggccgctcc caggaggact tcgtcaacga ccgccgccgc gtgcgcaggg 1440 actacgacgagttcaaggcg cgcatcaacg ggctggagaa cgacatcagg cagcgctccg 1500 acgcctacaacgccgccagg gggctcaagg acggcgagcc cagggctacg tggatggccg 1560 acggcacacagtgggagggc acctgggttg agccgtccga gaaccaccgc aagggcgacc 1620 atgccggcatcgtcctggtg cttctgaacc acccgagcca cagccgtcag ctcgggccgc 1680 cggcgagcgcggacaacccg ctggacttga gcatggtgga cgtgcggctc cccatgctgg 1740 tgtacgtctcccgcgagaag cggcccgggc acaaccacca gaagaaggcc ggcgccatga 1800 acgcgctgacccggtgctcc gccgtgctct ccaactcgcc cttcatcctg aacctggact 1860 gcgaccactacatcaacaac tcgcaggcgc tgcgcgcggg catctgcttc atgctcgggc 1920 gggacagcgacacggtggcg ttcgtccagt tcccgcagcg cttcgagggc gtggacccca 1980 cggacctgtacgccaaccac aaccgcatct tcttcgacgg cacgctccgg gcgctggacg 2040 gcatgcagggccccatctac gtcggcacgg gctgcctgtt ccgccgcatc acgctctacg 2100 gcttcgacccgccgcggatc aacgtgggcg ggccgtgctt cccgtcgctg ggcggcatgt 2160 tcgccaagaccaagtacgag aagcctgggc tggagctcac caccaaggcc gccgtggcca 2220 agggcaagcacggcttcctg cccatgccca agaagtcgta cggcaagtcg gacgcgttcg 2280 cggacaccatcccgatggcg tcgcacccgt cgccgttc 2318 30 590 PRT Zea mays 30 Met Ala ProGly Gly Arg Arg Ser Asn Gly Glu Thr Pro Thr Gly Gln 1 5 10 15 Gln GlnGln Gln Gln Gln Ala Asp Gly Arg Arg Gly Cys Ala Cys Gly 20 25 30 Gly PhePro Val Cys Ala Cys Ala Gly Ala Ala Ala Val Ala Ser Ala 35 40 45 Ala SerSer Ala Asp Met Asp Arg Val Ala Val Ala Ala Thr Glu Gly 50 55 60 Gln IleGly Ala Val Asn Asp Glu Ser Trp Val Ala Val Asp Leu Ser 65 70 75 80 AspAsp Gly Leu Ser Ser Ala Ala Asp Pro Gly Ala Val Ala Leu Glu 85 90 95 GluArg Pro Val Phe Arg Thr Glu Lys Ile Lys Gly Val Leu Leu His 100 105 110Pro Tyr Arg Val Leu Ile Phe Val Arg Leu Ile Ala Phe Thr Leu Phe 115 120125 Val Ile Trp Arg Ile Ser His Arg Asn Pro Asp Ala Leu Trp Leu Trp 130135 140 Val Thr Ser Ile Ala Gly Glu Phe Trp Phe Gly Phe Ser Trp Leu Leu145 150 155 160 Asp Gln Leu Pro Lys Leu Asn Pro Ile Asn Arg Val Pro AspLeu Gly 165 170 175 Ala Leu Arg Gln Arg Phe Asp Arg Ala Asp Gly Thr SerArg Leu Pro 180 185 190 Gly Leu Asp Ile Phe Val Thr Thr Ala Asp Pro PheLys Glu Pro Ile 195 200 205 Leu Ser Thr Ala Asn Ser Ile Leu Ser Ile LeuAla Ala Asp Tyr Pro 210 215 220 Val Glu Arg Asn Thr Cys Tyr Leu Ser AspAsp Ser Gly Met Leu Leu 225 230 235 240 Thr Tyr Glu Ala Met Ala Glu AlaAla Lys Phe Ala Thr Val Trp Val 245 250 255 Pro Phe Cys Arg Lys His GlyIle Glu Pro Arg Gly Pro Glu Ser Tyr 260 265 270 Phe Glu Leu Lys Ser HisPro Tyr Met Gly Arg Ser Gln Glu Asp Phe 275 280 285 Val Asn Asp Arg ArgArg Val Arg Arg Asp Tyr Asp Glu Phe Lys Ala 290 295 300 Arg Ile Asn GlyLeu Glu Asn Asp Ile Arg Gln Arg Ser Asp Ala Tyr 305 310 315 320 Asn AlaAla Arg Gly Leu Lys Asp Gly Glu Pro Arg Ala Thr Trp Met 325 330 335 AlaAsp Gly Thr Gln Trp Glu Gly Thr Trp Val Glu Pro Ser Glu Asn 340 345 350His Arg Lys Gly Asp His Ala Gly Ile Val Leu Val Leu Leu Asn His 355 360365 Pro Ser His Ser Arg Gln Leu Gly Pro Pro Ala Ser Ala Asp Asn Pro 370375 380 Leu Asp Leu Ser Met Val Asp Val Arg Leu Pro Met Leu Val Tyr Val385 390 395 400 Ser Arg Glu Lys Arg Pro Gly His Asn His Gln Lys Lys AlaGly Ala 405 410 415 Met Asn Ala Leu Thr Arg Cys Ser Ala Val Leu Ser AsnSer Pro Phe 420 425 430 Ile Leu Asn Leu Asp Cys Asp His Tyr Ile Asn AsnSer Gln Ala Leu 435 440 445 Arg Ala Gly Ile Cys Phe Met Leu Gly Arg AspSer Asp Thr Val Ala 450 455 460 Phe Val Gln Phe Pro Gln Arg Phe Glu GlyVal Asp Pro Thr Asp Leu 465 470 475 480 Tyr Ala Asn His Asn Arg Ile PhePhe Asp Gly Thr Leu Arg Ala Leu 485 490 495 Asp Gly Met Gln Gly Pro IleTyr Val Gly Thr Gly Cys Leu Phe Arg 500 505 510 Arg Ile Thr Leu Tyr GlyPhe Asp Pro Pro Arg Ile Asn Val Gly Gly 515 520 525 Pro Cys Phe Pro SerLeu Gly Gly Met Phe Ala Lys Thr Lys Tyr Glu 530 535 540 Lys Pro Gly LeuGlu Leu Thr Thr Lys Ala Ala Val Ala Lys Gly Lys 545 550 555 560 His GlyPhe Leu Pro Met Pro Lys Lys Ser Tyr Gly Lys Ser Asp Ala 565 570 575 PheAla Asp Thr Ile Pro Met Ala Ser His Pro Ser Pro Phe 580 585 590

1. An isolated nucleic acid molecule comprising a nucleotide sequenceselected from the group consisting of: a) a nucleotide sequencecomprising the sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, or 29; b) a nucleotide sequence comprisingthe cDNA insert of the plasmid deposited as Patent Deposit No. PTA-3610,PTA-3612, PTA-3611, or PTA-3613; c) a nucleotide sequence encoding apolypeptide comprising the amino acid sequence set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30; d) anucleotide sequence comprising at least 30 contiguous nucleotides of asequence of a), b), or c); e) a nucleotide sequence having at least 80%sequence identity to a sequence of a), b), or c), wherein said sequenceencodes a polypeptide that retains polysaccharide synthase activity; f)a nucleotide sequence comprising an antisense sequence corresponding toa), b), c), d) or e); and g) a nucleotide sequence that hybridizes understringent conditions to a sequence of a), b), c), d), e), f), or acomplement thereof, wherein said sequence encodes a polypeptide thatretains polysaccharide synthase activity.
 2. An expression cassettecomprising a nucleotide sequence of 1, wherein said nucleotide sequenceis operably linked to a promoter that drives expression in a plant cell.3. The expression cassette of 2, wherein said promoter is a constitutivepromoter.
 4. A nucleotide construct comprising the expression cassetteof
 2. 5. A plant cell having stably incorporated into its genome anucleotide sequence operably linked to a promoter capable of initiatingtranscription in said plant cell, wherein said nucleotide sequence isselected from the group consisting of: a) a nucleotide sequencecomprising the sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, or 29; b) a nucleotide sequence comprisingthe cDNA insert of the plasmid deposited as Patent Deposit No. PTA-3610,PTA-3612, PTA-3611, or PTA-3613; c) a nucleotide sequence encoding apolypeptide comprising the amino acid sequence set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30; d) anucleotide sequence comprising at least 30 contiguous nucleotides of asequence of a), b), or c); e) a nucleotide sequence having at least 80%sequence identity to a sequence of a), b), or c), wherein said sequenceencodes a polypeptide that retains polysaccharide synthase activity; f)a nucleotide sequence comprising an antisense sequence corresponding toa sequence of a), b), c), d) or e); and g) a nucleotide sequence thathybridizes under stringent conditions to a sequence of a), b), c), d),e), f), or a complement thereof, wherein said sequence encodes apolypeptide that retains polysaccharide synthase activity.
 6. The plantcell of 5, wherein said plant cell is from a monocotyledonous plant. 7.The plant cell of 5, wherein said plant cell is from a dicotyledonousplant.
 8. A plant having stably incorporated in its genome a nucleotidesequence operably linked to a heterologous promoter capable ofinitiating transcription in said plant, wherein said nucleotide sequenceis selected from the group consisting of: a) a nucleotide sequencecomprising the sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, or 29; b) a nucleotide sequence comprisingthe cDNA insert of the plasmid deposited as Patent Deposit No. PTA-3610,PTA-3612, PTA-3611, or PTA-3613; c) a nucleotide sequence encoding apolypeptide comprising the amino acid sequence set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30; d) anucleotide sequence comprising at least 30 contiguous nucleotides of asequence of a), b), or c); e) a nucleotide sequence having at least 80%sequence identity to a sequence of a), b), or c), wherein said sequenceencodes a polypeptide that retains polysaccharide synthase activity; f)a nucleotide sequence comprising an antisense sequence corresponding toa sequence of a), b), c), d) or e); and g) a nucleotide sequence thathybridizes under stringent conditions to a sequence of a), b), c), d),e), f), or a complement thereof, wherein said sequence encodes apolypeptide that retains polysaccharide synthase activity.
 9. The plantof 8, wherein said plant is a dicot.
 10. The plant of 8, wherein saidplant is a monocot.
 11. Transformed seed of a plant of any one of 8-10.12. An isolated polypeptide selected from the group consisting of: a) apolypeptide comprising the amino acid sequence set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30; b) apolypeptide encoded by the nucleic acid sequence of the cDNA insert ofthe plasmid deposited as Patent Deposit No. PTA-3610, PTA-3612,PTA-3611, or PTA-3613; c) a polypeptide encoded by a nucleotide sequencethat hybridizes under stringent conditions to a nucleotide sequencecomprising the sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, or 29, wherein said polypeptide retainspolysaccharide synthase activity; d) a polypeptide having at least 80%sequence identity to a polypeptide of a), b), or c), wherein saidpolypeptide retains polysaccharide synthase activity; and e) apolypeptide comprising at least 40 contiguous amino acids of thesequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, or
 30. 13. A method for modulating the level of apolysaccharide synthase in a plant, said method comprising stablytransforming a plant cell with a nucleotide sequence operably linked toa heterologous promoter capable of initiating transcription in a plant,and regenerating a transformed plant, wherein said nucleotide sequencecomprises a nucleotide sequence selected from the group consisting of:a) a nucleotide sequence comprising the sequence set forth in SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, or 29; b) anucleotide sequence comprising the cDNA insert of the plasmid depositedas Patent Deposit No. PTA-3610, PTA-3612, PTA-3611, or PTA-3613; c) anucleotide sequence encoding a polypeptide comprising the amino acidsequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, or 30; d) a nucleotide sequence comprising at least 30contiguous nucleotides of a sequence of a), b), or c); e) a nucleotidesequence having at least 80% sequence identity to a sequence of a), b),or c), wherein said sequence encodes a polypeptide that retainspolysaccharide synthase activity; f) a nucleotide sequence comprising anantisense sequence corresponding to a sequence of a), b), c), d) or e);and g) a nucleotide sequence that hybridizes under stringent conditionsto a sequence of a), b), c), d), e), f), or a complement thereof,wherein said sequence encodes a polypeptide that retains polysaccharidesynthase activity.
 14. The method of 13, wherein said plant is a dicot.15. The method of 14, wherein said dicot is soybean, sunflower, canola,alfalfa, cotton, Arabidopsis thaliana, tomato, Brassica vegetables,peppers, potatoes, apples, spinach, or lettuce.
 16. The method of 13,wherein said plant is a monocot.
 17. The method of 16, wherein saidmonocot is corn, sorghum, wheat, rice, barley, or millet.
 18. The methodof 13, wherein polysaccharide synthase is increased.
 19. The method of13, wherein polysaccharide synthase is decreased.
 20. A method formodulating the level of hemicellulose in a plant, said method comprisingstably transforming a plant cell with a nucleotide sequence operablylinked to a promoter capable of initiating transcription in a plant, andregenerating a transformed plant, wherein said nucleotide sequencecomprises a nucleotide sequence selected from the group consisting of:a) a nucleotide sequence comprising the sequence set forth in SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, or 29; b) anucleotide sequence comprising the cDNA insert of the plasmid depositedas Patent Deposit No. PTA-3610, PTA-3612, PTA-3611, or PTA-3613; c) anucleotide sequence encoding a polypeptide comprising the amino acidsequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, or 30; d) a nucleotide sequence comprising at least 30contiguous nucleotides of a sequence of a), b), or c); e) a nucleotidesequence having at least 80% sequence identity to a sequence of a), b),or c), wherein said sequence encodes a polypeptide that retainspolysaccharide synthase activity; f) a nucleotide sequence comprising anantisense sequence corresponding to a sequence of a), b), c), d) or e);and g) a nucleotide sequence that hybridizes under stringent conditionsto a sequence of a), b), c), d), e), f), or a complement thereof,wherein said sequence encodes a polypeptide that retains polysaccharidesynthase activity.
 21. The method of 20, wherein said hemicellulose isincreased.
 22. The method of 20, wherein said hemicellulose isdecreased.
 23. The method of 20, wherein said hemicellulose is selectedfrom the group consisting of arabinoxylans, xyloglucans, mixed-linkglucans, glucuronoxylans, and glucomannans.
 24. The method of 20,wherein said plant is a monocot.
 25. The method of 20, wherein saidplant is a dicot.
 26. A method for modulating the level of pectin in aplant, said method comprising stably transforming a plant cell with anucleotide sequence operably linked to a promoter capable of initiatingtranscription in a plant, and regenerating a transformed plant, whereinsaid nucleotide sequence comprises a nucleotide sequence selected fromthe group consisting of: a) a nucleotide sequence comprising thesequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, or 29; b) a nucleotide sequence comprising the cDNA insertof the plasmid deposited as Patent Deposit No. PTA-3610, PTA-3612,PTA-3611, or PTA-3613; c) a nucleotide sequence encoding a polypeptidecomprising the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30; d) a nucleotide sequencecomprising at least 30 contiguous nucleotides of a sequence of a), b),or c); e) a nucleotide sequence having at least 80% sequence identity toa sequence of a), b), or c), wherein said sequence encodes a polypeptidethat retains polysaccharide synthase activity; f) a nucleotide sequencecomprising an antisense sequence corresponding to a sequence of a), b),c), d) or e); and g) a nucleotide sequence that hybridizes understringent conditions to a sequence of a), b), c), d), e), f), or acomplement thereof, wherein said sequence encodes a polypeptide thatretains polysaccharide synthase activity.
 27. The method of 26, whereinpectin is increased.
 28. The method of 26, wherein pectin is decreased.29. The method of 26, wherein said pectin is selected from the groupconsisting of polygalacturonans, rhamnogalacturonans, arabinogalactans,arabinans, and galactans.
 30. The method of 26, wherein said plant is amonocot.
 31. The method of 26, wherein said plant is a dicot.
 32. Amethod for improving the digestibility of a crop, said method comprisingstably transforming a plant cell with a nucleotide sequence operablylinked to a promoter capable of initiating transcription in a plant, andregenerating a transformed plant wherein said nucleotide sequencecomprises a nucleotide sequence selected from the group consisting of:a) a nucleotide sequence comprising the sequence set forth in SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, or 29; b) anucleotide sequence comprising the cDNA insert of the plasmid depositedas Patent Deposit No. PTA-3610, PTA-3612, PTA-3611, or PTA-3613; c) anucleotide sequence encoding a polypeptide comprising the amino acidsequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, or 30; d) a nucleotide sequence comprising at least 30contiguous nucleotides of a sequence of a), b), or c); e) a nucleotidesequence having at least 80% sequence identity to a sequence of a), b),or c), wherein said sequence encodes a polypeptide that retainspolysaccharide synthase activity; f) a nucleotide sequence comprising anantisense sequence corresponding to a sequence of a), b), c), d) or e);and g) a nucleotide sequence that hybridizes under stringent conditionsto a sequence of a), b), c), d), e), f), or a complement thereof,wherein said sequence encodes a polypeptide that retains polysaccharidesynthase activity.
 33. A method for modulating gum extraction fromcrops, said method comprising stably transforming a plant cell with anucleotide sequence operably linked to a promoter capable of initiatingtranscription in a plant, wherein said nucleotide sequence comprises anucleotide sequence selected from the group consisting of: a) anucleotide sequence comprising the sequence set forth in SEQ ID NO: 1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, or 29; b) a nucleotidesequence comprising the cDNA insert of the plasmid deposited as PatentDeposit No. PTA-3610, PTA-3612, PTA-3611, or PTA-3613; c) a nucleotidesequence encoding a polypeptide comprising the amino acid sequence setforth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,or 30; d) a nucleotide sequence comprising at least 30 contiguousnucleotides of a sequence of a), b), or c); e) a nucleotide sequencehaving at least 80% sequence identity to a sequence of a), b), or c),wherein said sequence encodes a polypeptide that retains polysaccharidesynthase activity; and f) a nucleotide sequence that hybridizes understringent conditions to a sequence of a), b), c), d), e), f), or acomplement thereof, wherein said sequence encodes a polypeptide thatretains polysaccharide synthase activity.
 34. A method for modulatinggrowth rate of a plant, said method comprising stably transforming aplant cell with a nucleotide sequence operably linked to a promotercapable of initiating transcription in a plant, wherein said nucleotidesequence comprises a nucleotide sequence selected from the groupconsisting of: a) a nucleotide sequence comprising the sequence setforth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,or 29; b) a nucleotide sequence comprising the cDNA insert of theplasmid deposited as Patent Deposit No. PTA-3610, PTA-3612, PTA-3611, orPTA-3613; c) a nucleotide sequence encoding a polypeptide comprising theamino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, or 30; d) a nucleotide sequence comprising atleast 30 contiguous nucleotides of a sequence of a), b), or c); e) anucleotide sequence having at least 80% sequence identity to a sequenceof a), b), or c), wherein said sequence encodes a polypeptide thatretains polysaccharide synthase activity; f) a nucleotide sequencecomprising an antisense sequence corresponding to a sequence of a), b),c), d) or e); and g) a nucleotide sequence that hybridizes understringent conditions to a sequence of a), b), c), d), e), f), or acomplement thereof, wherein said sequence encodes a polypeptide thatretains polysaccharide synthase activity.